Production of chimeric antibodies - a combinatorial approach

ABSTRACT

Methods are disclosed which may be used for the production of antibodies, or antibody fragments, which have the same binding specificity as a parent antibody but which have increased human characteristics. Humanized antibodies may be obtained by chain shuffling, perhaps using phage display technology. In one embodiment, a polypeptide comprising a heavy or light chain variable domain of a non-human antibody specific for an antigen of interest is combined with a repertoire of human complementary (light or heavy) chain variable domains. Hybrid pairings which are specific for the antigen of interest are selected. Human chains from the selected pairings may then be combined with a repertoire of human complementary variable domains (heavy or light) and humanized antibody polypeptide dimers can then be selected for binding specificity for antigen. The methods may be combined with CDR-imprinting. In another embodiment, component part of an antigen-binding site of a no-human antibody known to bind a particular antigen is combined with a repertoire of component parts of an antigen-binding site of human antibody, forming a library of antibody polypeptide dimers with antigen-binding sites. Hybrids selected from this library may be used in a second humanizing shuffling step, or may already be of sufficient human character to be of value, perhaps after some modification to increase human character still further.

The present invention relates to the production of antibodies. Moreparticularly, it relates to the production of antibodies with increasedhuman characteristics over a parent antibody specific for the sameantigen.

Many rodent monoclonal antibodies have been isolated using hybridomatechnology and utilised for in vivo therapeutic and diagnostic purposesin humans. For example, an early application of these mouse monoclonalantibodies was as targeting agents to kill or image tumours (F. H.Deland and D. M. Goldenberg 1982 in `Radionuclide Imaging` ed. D. E.Kuhl pp289-297, Pergamon, Paris; R. Levy and R. A. Miller Ann. Rev. Med.1983, 34 pp107-116). However, the use of such antibodies in vivo canlead to problems. The foreign immunoglobulins can elicit ananti-globulin response (known as a human anti-mouse antibody (HAMA)response) Which can interfere with therapy (R. A. Miller et al, 1983Blood 62 988-995) or cause allergic or immune complex hypersensitivity(B. Ratner, 1943, Allergy, Anaphylaxis and Immunotherapy Williams andWilkins, Baltimore).

To overcome these problems, Winter and colleagues (GB2188638B) developeda method of humanising or "reshaping` such antibodies. Thecomplementarity determining regions (CDRs) of the mouse antibody, whichcomprise the antigen combining site, are inserted into human frameworkregions thereby generating antibodies in which only the CDR sequencesare derived from the original mouse antibody. This is the techniqueknown as "CDR-grafting" or "CDR-imprinting". One such reshaped antibodyCAMPATH-1 (L. Riechmann et al, 1988 Nature 332, pp323-327 has been usedsuccessfully in the treatment of B cell lymphoma (G. Hale et al, 1988Lancet 2, pp1394-1399) and vasculitis (P. W. Mathieson et al New Engl.J. Med. 1990 323, pp250-254) and rheumatoid arthritis (V. Kyle et at1991 J. Rheumatol. 18, pp1737-1738). This has prompted the humanisationof a large number of antibodies for therapeutic purposes directedagainst cancer markers, for example the interleukin 2 receptor (C. Queenet al, 1989 Proc. Natl. Acad. Sci. USA, 86, pp10029-10033); epidermalgrowth factor receptor (C. A. Kettleborough et al 1991 Protein Eng. 4,pp773-783; P. Carter et al 1992 Proc. Natl. Acad. Sci. USA 89,pp4285-4289) and carcinoembryonic antigen (K. Bosslet et al. Brit. J.Cancer 65, pp234-238, 1992). A number of antibodies directed againstinfectious viruses have also been humanised, for instance antibodiesdirected against respiratory syncytial virus (P. R. Tempest et al, 1991Bio/Technology 9, pp266-271); herpes simplex virus (M. S. Co et al 1991Proc. Natl. Acad. Sci. USA 88, pp2869-2873) and human immunodeficiencyvirus (H. Maeda et al 1991 Human Antibodies and Hybridomas 2,pp124-134). Humanised antibodies have also been used for imaging tumoursafter labelling with radioisotopes (V. Hird et al, 1991 Brit. J. Cancer64 911-914).

Successful reshaping depends on the rodent and human framework regionsbeing structurally conserved both in the orientation of the beta-sheetsof each domain and in the packing of the heavy and light chainstogether; the hypervariable loops making the majority of contacts withantigen and the loops being supported in a similar way by the underlyingbeta-sheet framework. Although these conditions are likely to be truefor some antibodies, the restitution of key contacts between the loopsand the framework has proved necessary in others, and has been assistedby molecular modelling (Riechmann et al, 1988 supra; Tempest et al, 1991supra) and systematic matching of rodent and human framework regions tominimise differences in primary sequences (Queen et al, 1989 supra;Gorman et al, 1991 supra; Maeda et al supra). Studies have shown thatthere are a number of residues in the `Vernier` zone underlying the CDRsof both heavy and light chain variable domains which may adjust CDRstructure and fine tune to fit the antigen and thus have a strong effecton affinity and specificity (J. Foote and G. Winter 1992 J. Mol. Biol.224 487-499).A variation of this approach is to transfer the mouse CDRsonto a chimaeric human/mouse framework in which the buried amine acidresidues are derived from the mouse antibody and the exposed amine acidsare derived from homologous human frameworks (E. A. Padlan et al1991Mol. Immunol. 28, 485-498).

The process of CDR grafting involves the transfer of the antigen bindingsite from a non-human (animal) antibody to the human antibody. In mostcases all three CDRs from both heavy and light chains have beentransplanted from the animal antibody to a single human framework. It isexpected that it should not always be necessary to transplant all theCDRs, as some CDRs may not be involved in binding to antigen, and CDRswith different sequences (and the same length) can have the same folding(and therefore contacts from antigen to the main chain contacts could beretained despite the different sequences). Indeed single domains (Wardet al, 1989, Nature 341, pp.544-546) or even single CDRs (R. Taub et al,1989, J. Biol Chem 264, pp.259-265) can have antigen binding activitiesalone. However, whether all or only some of the CDRs are transplanted,the intention of CDR grafting is to transplant the same, or much thesame antigen binding site, from animal to human antibodies.

Structurally, the simplest antibody (IgG) comprises four polypeptidechains, two heavy (H) chains and two light (L) chains inter-connected bydisulphide bonds (see FIG. 1). The light chains exist in two distinctforms called kappa (K) and lambda (λ). Each chain has a constant region(C) and a variable region (V). Each chain is organized into a series ofdomains. The light chains have two domains, corresponding to the Cregion and the other to the V region. The heavy chains have fourdomains, one corresponding to the V region and three domains (1, 2 and3) in the C region. The antibody has two arms (each arm being a Fabregion), each of which has a VL and a VH region associated with eachother. It is this pair of V regions (VL and VH) that differ from oneantibody to another (owing to amino acid sequence variations), and whichtogether are responsible for recognising the antigen and providing anantigen binding site (ABS). In even more detail, each V region is madeup from three complementarity determining regions (CDR) separated byfour framework regions (FR). The CDR's are the most variable part of thevariable regions, and they perform critical antigen binding function.The CDR regions are derived from many potential germ line sequences viaa complex process involving recombination, mutation and selection.

It has been shown that the function of binding antigens can be performedby fragments of a whole antibody. Example binding fragments are (i) theFab fragment consisting of the VL, VH, CL and CH1 domains; (ii) the Fdfragment consisting of the VH and CH1 domains; (iii) the Fv fragmentconsisting of the VL and VH domains of a single arm of an antibody, (iv)the dab fragment (Ward, E. S. et al., Nature 341, 544-546 (1989) whichconsists of a VH domain; (v) isolated CDR regions; and (vi) F(ab')₂fragments, a bivalent fragment comprising two Fab fragments linked by adisulphide bridge at the hinge region.

Although the two domains of the Fv fragment are coded for by separategenes, it has proved possible to make a synthetic linker that enablesthem to be made as a single protein chain (known as single chain Fv(scFv); Bird, R. E. et al., Science 242, 423-426 (1988) Huston, J. S. etal., Proc. Natl. Acad. Sci., USA 85, 5879-5883 (1988)) by recombinantmethods.

Experience with humanisation of monoclonal antibodies has shown thatthese molecules can be of great value in therapy and diagnosis. However,there is a requirement to be able to monitor a number of humanisedantibody derivative molecules to obtain one which retains or improves onthe original characteristics of the mouse monoclonals. Further there isa requirement to generate such molecules by a rapid and convenientprocedure.

TERMINOLOGY

Much of the terminology discussed in this section has been mentioned inthe text where appropriate.

Replicable Genetic Display Package (Rgdp)

This describes a biological particle which has genetic informationproviding the particle with the ability to replicate. The particle candisplay on its surface at least part of a polypeptide. The polypeptidecan be encoded by genetic information native to the particle and/orartificially placed into the particle or an ancestor of it. Thedisplayed polypeptide may be any member of a specific binding pair eg.heavy or light chain domains based on an immunoglobulin molecule, anenzyme or a receptor etc.

The particle may be a virus eg. a bacteriophage such as fd or M13.

Package

This describes a replicable genetic display package in which theparticle is displaying a member of a specific binding pair at itssurface. The package may be a bacteriophage which displays an antigenbinding domain at its surface. This type of package has been called aphage antibody (pAb).

Antibody

This describes an immunoglobulin whether natural or partly or whollysynthetically produced. The term also covers any protein having abinding domain which is homologous to an immunoglobulin binding domain.These proteins can be derived from natural sources, or partly or whollysynthetically produced.

Example antibodies are the immunoglobulin isotypes and the Fab, F(ab¹)₂,scFv, Fv, dab, Fd fragments.

Antibody Polypeptide Dimer

An antibody polypeptide dimer is an association of two polypeptide chaincomponents of an antibody, capable of binding an antigen. Thus, it maybe one arm of an antibody consisting of a heavy chain and a light chain,it may be a Fab fragment consisting of V_(L), V_(H), C_(L) and C_(H) 1antibody domains, an Fv fragment consisting of a V_(L) domain and aV_(H) domain, or a scFv ("single chain Fv") fragment consisting of aV_(L) domain linked to a V_(H) domain by a synthetic linker. An scFvfragment is a single polypeptide chain that falls within a definition ofan "antibody polypeptide dimer" because it consists of two polypeptidechain components of an antibody, associated by means of the syntheticlinker, and is capable of binding an antigen.

Immunoglobulin Superfamily

This describes a family of polypeptides, the members of which have atleast one domain with a structure related to that of the variable orconstant domain of immunoglobulin molecules. The domain contains twoB-sheets and usually a conserved disulphide bond (see A. F. Williams andA. N. Barclay 1988 Ann. Rev Immunol. 6 381-405).

Example members of an immunoglobulin superfamily are CD4, plateletderived growth factor receptor (PDGFR), intercellular adhesion molecule.(ICAM). Except where the context otherwise dictates, reference toimmunoglobulins and immunoglobulin homologs in this application includesmembers of the immunoglobulin superfamily and homologs thereof.

Homologs

This term indicates polypeptides having the same or conserved residuesat a corresponding position in their primary, secondary or tertiarystructure. The term also extends to two or more nucleotide sequencesencoding the homologous polypeptides.

Example homologous peptides are the immunoglobulin isotypes.

Genetically diverse population

In connection with antibodies or polypeptide components thereof, this isreferring not only to diversity that can exist in the natural populationof cells or organisms, but also diversity that can be created byartificial mutation in vitro or in vivo.

Mutation in vitro may for example, involve random mutagenesis usingoligonucleotides having random mutations of the sequence desired to bevaried. In vivo mutagenesis may for example, use mutator strains of hostmicroorganisms to harbour the DNA (see Example 38 of WO 92/01047). Thewords "unique population" may be used to denote a plurality of e.g.polypeptide chains, which are not genetically diverse i.e. they are allthe same. A restricted population is one which is diverse but less sothan the full repertoire of an animal. The diversity may have beenreduced by prior selection, eg using antigen binding specificity.

Domain

A domain is a part of a protein that is folded within itself andindependently of other parts of the same protein and independently of acomplementary binding member.

Folded Unit

This is a specific combination of an alpha-helix and/or beta-strandand/or beta-turn structure. Domains and folded units contain structuresthat bring together amino acids that are not adjacent in the primarystructure.

Free Form

This describes the stare of a polypeptide which is not displayed by areplicable genetic display package.

Conditionally Defective

This describes a gene which does not express a particular polypeptideunder one set of conditions, but expresses it under another set ofconditions. An example, is a gene containing an amber mutation expressedin non-suppressing or suppressing hosts respectively.

Alternatively, a gene may express a protein which is defective under oneset of conditions, but not under another set. An example is a gene witha temperature sensitive mutation.

Suppressible Translational Stop Codon

This describes a codon which allows the translation of nucleotidesequences downstream of the codon under one set of conditions, but underanother set of conditions translation ends at the codon. Example ofsuppressible translational stop codons are the amber, ochre and opalcodons.

Mutator Strain

This is a host cell which has a genetic defect which causes DNAreplicated within it to be mutated with respect to its parent DNA.Example mutator strains are NR9046mutD5 and NR9046 mut T1 (See Example38 of WO 92/01047).

Helper Phage

This is a phage which is used to infect cells containing a defectivephage genome and which functions to complement the defect. The defectivephage genome can be a phagemid or a phage with some function encodinggene sequences removed. Examples of helper phages are M13KO7, M13K07gene III no. 3; and phage displaying or encoding a binding moleculefused to a capsid protein.

Vector

This is a DNA molecule, capable of replication in a host organism, intowhich a gene is inserted to construct a recombinant DNA molecule.

Phage Vector

This is a vector derived by modification of a phage genome, containingan origin of replication for a bacteriophage, but not one for a plasmid.

Phagemid Vector

This is a vector derived by modification of a plasmid genome, containingan origin of replication for a bacteriophage as well as the plasmidorigin of replication.

Secreted

This describes a rgdp or molecule that associates with the polypeptidedisplayed on the rgdp, in which the polypeptide and/or the molecule,have been folded and the package assembled externally to the cellularcytosol.

Repertoire of Rearranged Immunoglobulin Genes

A collection of naturally occurring nucleotides eg DNA sequences whichencoded expressed immunoglobulin genes in an animal. The sequences aregenerated by the in vivo rearrangement of eg V, D and J segments for Hchains and eg the V and J segments for L chains. Alternatively thesequences may be generated from a cell line immunised in vitro and inwhich the rearrangement in response to immunisation occursintracellularly. The word "repertoire" is used to indicate geneticdiversity.

Library

A collection of nucleotide eg DNA, sequences within clones; or agenetically diverse collection of polypeptides, or specific binding pairmembers, or polypeptides or sbp members displayed on rgdps capable ofselection or screening to provide an individual polypeptide or sbpmembers or a mixed population of polypeptides or sbp members.

Repertoire of Artificially Rearranged Immunoglobulin Genes

A collection of nucleotide eg DNA, sequences derived wholly or partlyfrom a source other than the rearranged immunoglobulin sequences from ananimal. This may include for example, DNA sequences encoding VH domainsby combining unrearranged V segments with D and J segments and DNAsequences encoding VL domains by combining V and J segments.

Part or all of the DNA sequences may be derived by oligonucleotidesynthesis.

Secretory Leader Peptide

This is a sequence of amine acids joined to the N-terminal end of apolypeptide and which directs movement of the polypeptide out of thecytosol.

Eluant

This is a solution used to breakdown the linkage between two molecules.The linkage can be a non-covalent or covalent bond(s). The two moleculescan be members of a sbp.

Derivative

This is a substance which derived from a polypeptide which is encoded bythe DNA within a selected rgdp. The derivative polypeptide may differfrom the encoded polypeptide by the addition, deletion, substitution orinsertion of amine acids, or by the linkage of other molecules to theencoded polypetide. These changes may be made at the nucleotide orprotein level. For example the encoded polypeptide may be a Fab fragmentwhich is then linked to an Fc tail from another source. Alternativelymarkers such as enzymes, flouresceins etc may be linked to eg Fab, scFvfragments.

According to one aspect of the present invention there is provided amethod of producing antibody polypeptide dimers specific for an antigenof interest, the method having the following steps:

(i) providing nucleic acid expression vectors which are capable of beingpackaged using a component of a replicable genetic display package(rgdp);

(ii) combining (a) a genetically diverse repertoire of nucleic acidsequences which each encode a first component part of an antigen-bindingsite of a human antibody with (b) nucleic acid which encodes a unique orgenetically diverse population of a second component part of anantigen-binding site of a non-human antibody known to bind said antigenof interest, to form a library of nucleic acid sequences on saidexpression vectors encoding antibody polypeptide dimers, which dimerseach consist of a first polypeptide chain component and a secondpolypeptide chain component, a first antigen-binding site component partand a second antigen-binding site component part in combination formingan antigen-binding site of an antibody polypeptide dimer;

(iii) expressing said library from said vectors in recombinant hostorganism cells, each of the said first polypeptide chain componentsbeing expressed as a fusion with a component of an rgdp which therebydisplays said first polypeptide chain component at the surface of rgdps;

(iv) selecting from said expressed library by binding with antigen aunique or restricted population of said antibody polypeptide dimerswhich have binding specificity for said antigen of interest, eachselected antibody polypeptide dimer being associated in its respectivergdp with nucleic acid encoding a said first component part of theantigen-binding site thereof.

Antibody polypeptide dimers are defined elsewhere in the text. The termis broad enough to encompass Fab, Fv and scFv fragments of antibodies aswell as a complete arm of anantibody.

A "component part of an antibody antigen-binding site" may be orcorrespond to a polypeptide chain component, eg a VH or a VL domain.However, it may be a CDR, or a VL sequence plus CDR of a VH, a VHsequence plus CDR of a VL, a VH plus VL sequence lacking only a CDR, andso on. The proviso is that the first and second component parts of anantigen-binding site of an antibody must in combination (together) forman antigen-binding site. Thus, if the second component part of anantigen-binding site of a non-human antibody specific for an antigen ofinterest is a CDR, then the first component part of an antigen-bindingsite of a human antibody will comprise the remainder of a VH and VLregion required to form a antigen-binding site (with or withoutassociated antibody constant domains (in a Fab format), or with orwithout a linker peptide sequence (in a Fv format). (In a scFv format alinker peptide links a light chain domain to a heavy chain domain.) Thesecond component part of an antigen-binding site of a non-human antibodymay comprise a VL domain plus part of a VH domain, that part being oneor more CDRs,for instance, perhaps CDR3. In such case, the firstcomponent part of an antigen-binding site of a human antibody wouldcomprise the remainder of a VH sequence which in combination with thesecond component part forms an antigen-binding site. Of course, theconverse situation holds and the person skilled in the art will be ableto envisage other combinations of first and second component parts whichtogether form an antigen-binding site.

There may be an additional step (v) wherein antibody polypeptide dimersselected in step (iv) are modified to remove or reduce non-humancharacteristics. This modification may be by genetically altering thedimers or a component thereof. This genetic alteration may be mutation,point mutation, insertion or deletion, for instance, specifically toremove or mask non-human residues, particularly those residues whichmight invoke an anti-idiotypic immune response upon administration ofthe non-human/human hybrid antibody polypeptide dimers to a human.

The modification of step (v) may comprise:

(a) combining a unique or restricted population of nucleic acid encodingsaid first antigen-binding site component parts selected in step (iv)with a genetically diverse repertoire of nucleic acid sequences eachencoding a second component part of an antigen-binding site of humanantibody, to form a second library of nucleic acid sequences encodingantibody polypeptide dimers, each antibody polypeptide dimer comprisinga second antibody polypeptide chain component which is expressed fromnucleic acid which is capable of being packaged using a component of anrgdp, said second chain component being expressed as a fusion with acomponent of an rgdp which thereby display it at the surface of rgdps,so that antibody polypeptide dimers specific for said antigen ofinterest, are selectable from said second library by binding with saidantigen of interest. each antibody polypeptide dimer being associated inits rgdp with nucleic acid encoding a second polypeptide componentthereof.

Selected antibody polypeptide dimers may be expressed as solublepolypeptides or in free form.

As explained, said second component part of an antigen-binding site of anon-human antibody may be an antibody region which makes contact withantigen, that region perhaps being a complementarity determining region(CDR).

Said second component part of an antigen-binding site of a non-humanantibody may be a second polypeptide chain component of an antibody.

The sequence encoding each said second component part of non-humanantibody may be genetically altered to increase its hemology to a secondcomponent part of a human antibody prior to said combination in step(ii) of the method.

Each antibody polypeptide dimer of any of said libraries of antibodypolypeptide dimers may be expressed as a single polypeptide chain, whichcould be a scFv fragment (preferably). An scFv fragment meets therequirements of the definition of an antibody polypeptide dimer.

On the other hand, each antibody polypeptide dimer of any of saidlibraries of antibody polypeptide dimers may be expressed as twopolypeptide chains, preferably as a Fv or a Fab fragment, but alsoperhaps as polypeptides with additional, non-antibody domains which willinteract, either covalently or non-covalently, to hold the variabledomains in a conformation which allows them to form an antigen-bindingsite.

According to an aspect of the present invention there is provided amethod of producing human antibody polypeptide dimers specific for anantigen of interest, comprising

(i) combining a diverse population of polypeptides each comprising avariable domain of a first polypeptide chain of a human antibody(population A) with a unique or restricted population of polypeptideseach comprising a variable domain of a second polypeptide chain of anon-human antibody specific for said antigen (population B), therebyforming a library of antibody polypeptide dimers each consisting of apolypeptide which comprises a variable domain of a first polypeptidechain of a human antibody and a polypeptide which comprises a variabledomain of a second polypeptide chain of a non-human antibody;

(ii) selecting from said library a unique or restricted population ofsaid antibody polypeptide dimers which have binding specificity for saidantigen (population C);

(iii) combining a unique or restricted population of polypeptidesderived from polypeptide dimers selected in step (ii) each comprising ahuman first polypeptide chain (population D) with a diverse populationof polypeptides each comprising a variable domain of a secondpolypeptide chain of a human antibody (population E), thereby forming alibrary of human antibody polypeptide chain dimers from which a uniqueor restricted population of human antibody polypeptide dimers specificfor said antigen (population F) are selectable.

In a preferred embodiment of this aspect:

(a) said polypeptides of population A are each expressed from a vector(X) in recombinant host organism cells as a fusion with a component of areplicable genetic display package (rgdp) which thereby displays saidpolypeptide at the surface of rgdps in a first population of rgdps;

(b) nucleic acid of said vector (X) is capable of being packaged usingsaid rgdp component, whereby the genetic material of each said rgdpencodes a said polypeptide of population A, so that the antibodypolypeptide dimers of population C are each associated in theirrespective rgdp with nucleic acid encoding a polypeptide comprising avariable domain of a first polypeptide chain of a human antibody;

(c) each of said polypeptides of population E is expressed from a vector(Y) in recombinant host organism cells as a fusion with a component of argdp which thereby displays it at the surface of rgdps in a secondpopulation of rgdps; and

(d) nucleic acid of said vector (Y) is capable of being packaged usingsaid rgdp component, whereby the genetic material of each said rgdp inthe second population of rgdps encodes a said polypeptide of populationE.

Said population A may be expressed from the same vector as saidpopulation B, or these populations may be not expressed from the samevector. Said population D may be expressed from the same vector as saidpopulation E, or these populations D and E may be not expressed from thesame vector. In other words, the method might be in a dual combinatorialformat or not, perhaps being in a hierarchical format, as discussedelsewhere, amongst other possibilities.

Each antibody polypeptide dimer of any of said libraries of antibodypolypeptide dimers may be expressed as a single polypeptide chain, whichmay be a scFv fragment.

Each antibody polypeptide dimer of any of said libraries of antibodypolypeptide dimers may be expressed as two polypeptide chains,preferably a Fv or Fab fragment, as discussed already above in thecontext of another aspect of the present invention.

Polypeptides of said population B may be chimaeric, each comprising ahuman antibody constant domain (along with a non-human, eg. rodent,variable domain).

The polypeptides of said population E may each comprise a region from anon-human antibody specific for said antigen, which region is one whichmakes contact with antigen, such as a complementarity determining region(CDR), particularly CDR3.

Said first polypeptide chain may be antibody light chain, said secondpolypeptide chain being antibody heavy chain, or vice versa.

There may be an additional step of selecting said population F of humanantibody polypeptide dimers specific for said antigen.

Any one of said populations C and F may be selected by binding with saidantigen.

The present invention also encompasses kits comprising vectors andreagents for use in any of the methods. It further extends to librariesand antibodies or antibody fragments produced by any of the methods.Also encompassed by the present invention is a method wherein nucleicacid encoding any selected polypeptide components of an antibodypolypeptide dimer, or encoding a selected antibody polypeptide dimer, isused to express said polypeptide component or polypeptide dimer or aderivative thereof in a recombinant host organism. Any polypeptide ordimer from a library produced using a method according to the presentinvention may be modified to produce a derivative thereof, or used toprepare a therapeutic or prophylactic medicament or a diagnosticproduct.

Demonstrated here are new methods of making antibodies, by utilisingsequences from the antigen binding site of an antibody or population ofantibodies directed against an antigen of interest. This offers anothermeans of humanising animal antibodies with defined antigen bindingactivities. Thus for a single rodent antibody, sequences comprising partof the antigen binding site of the antibody may be combined with diverserepertoires of sequences of human antibodies that can in combinationcreate a complete antigen binding site. The original rodent (and human)sequences could comprise entire chains or parts of chains. For examplethe light chain of a rodent antibody could be combined with a repertoireof human heavy chain variable domains; or the third CDR of the heavychain of a rodent antibody could be combined with a repertoire of humanheavy chains encoding the rest of the heavy chain variable domains and arepertoire of human light chain variable domains. The antigen bindingsites comprising these sequences are then selected for binding toantigen.

The sequences comprising a component part of the antigen binding sitecould be a portion of primary sequence generally thought to be involvedin antigen binding, for example a CDR or CDRs, or the residues at thetip of the loop of (a) CDR(s). Alternatively, the sequences could besequences of the antibody that are known to contact the antigen, asdetermined for example from the structure of a complex of antigen andantibody solved by X-ray crystallography or NMR, or as determined fromsite directed mutagenesis of the antibody.

The antigen binding sites created by this process differ from thosecreated by CDR grafting, in that only the portion of sequence of theoriginal rodent antibody is likely to make contacts with antigen in asimilar manner. The selected human sequences are likely to differ insequence and make alternative contacts with the antigen from those ofthe original binding site. However, the constraints imposed by bindingof the portion of original sequence to antigen and the shapes of theantigen and its antigen binding sites, are likely to drive the newcontacts of the human sequences to the same region or epitope of theantigen. We have therefore termed the process "epitope imprintedselection" (EIS).

Starting with an animal antibody, one process results in the selectionof antibodies that are partly human antibodies. Such antibodies may besufficiently similar in sequence to human antibodies to be used directlyin therapy or after alteration of a few key residues. For example,antibodies created with human heavy and light chain variable domainsthat also comprise the VH-CDR3 region from a rodent antibody willclosely resemble genuine human antibodies as the CDR3 region of bothhuman and rodent antibodies is highly variable. Likewise the lightchains of some rodent antibodies are very similar in sequence to thelight chains of human anitbodies. Thus antibodies created from theserodent light chains in combination with a repertoire of human heavychains may closely resemble human antibodies. Sequence differencesbetween the rodent component of the selected antibody with humansequences could be minimised by replacing those residues that differwith the residues of human sequences, for example, by site directedmutagenesis of individual residues, or by CDR grafting of entire loops.Instead of starting with an animal antibody, it should also be possibleto start with an genetically engineered antibody derived from an animalantibody. The starting engineered antibody might for example be a CDRgrafted antibody with human framework regions and mouse complementarityregions (as described in GB2188638B, for instance). Alternatively thestarting engineered antibody could comprise heavy or light chains inwhich residues had been altered to maximise sequence homology with humanantibodies.

However, the invention may also be used to create antibodies withentirely human sequences. For example, starting with a partly humanantibody with human heavy chain non-human, e.g. mouse, light chain, thenon-human light chain could be replaced by repertoires of human lightchains. In this way, the sequences of the selected antibodies will beentirely human and yet the antigen binding site should be directedtowards the same epitope of the antigen. EIS therefore offers a methodfor making partly human or entirely human antibodies that bind to thesame epitope as animal or partly human antibodies respectively.

In CDR grafting the entire antigen binding site is transplanted, and itis usually necessary to test only one or a few antibodies for binding toantigen. However, EIS may use only part of the antigen binding site incombination with a, perhaps very large, repertoire of human sequences,when there is a requirement for the screening of a much larger number ofantibodies. Indeed in a preferred embodiment, repertoires of antibodyfragments are displayed on the surface of filamentous phase and thegenes encoding fragments with antigen binding activities are selected bybinding of the phase to antigen. It was disclosed in patent applicationWO 92/01047 that antibody fragments can be displayed on phase and thatthey will bind antigen and that they can be selected. The ability toselect antibodies provided by the use of display on phase as describedin WO 92/01047 allows the rapid screening of large numbers of humanisedantibody derivatives which can be generated using insertion of CDRs atrestriction sites or in vitro mutagenesis techniques (Riechmann et al,1988 supra; Patent GB2188638B) or using PCR technology (B. L. Daughertyet al 1991 Nucl. Acids Res. 19, pp2471-2476). In the application WO92/01047, the generation of hierarchical libraries was described inwhich one antibody heavy or light chain variable domain was keptconstant and displayed on phase with a library of the complementarychain variable domain for the selection of partners that bind toantigen. Such an approach generates a highly diverse pool of heavy andlight chain variable domain combinations from which antibodyspecificities can be selected. Chain shuffling has been applied, forexample, to obtaining further light chain and heavy chain partners forthe complementary chain of a hapten binding antibody (Clackson et al,1991, Nature, 352, 624-628) and to build high affinity human antibodies(J. D. Marks et al, 1992 Bio/Technology 10, 779-783). In these cases,the new partner chains were highly related in sequence to those chainsseen in the original combination.

In these examples, chains of different species were not combined.However, there is a report of an attempt to shuffle the heavy chains ofa mouse antibody directed against a hapten with a repertoire of humanlight chains (and also with a repertoire of mouse light chains), andscreening for binding of individual clones to antigen usingnitrocellulose filters. However, no binders were detected, nor was theprocess proposed as a means to "humanise" mouse antibodies (A. S. Kanget al, Proc. Natl. Acad. Sci. USA 1991, 88, 11120-11123). The authorsconcluded that the "redesign of antibodies through recombination of asomatically mutated chain with a naive partner may be a difficultprocess". The use of EIS (as illustrated by chain shuffling using chainsfrom different species) therefore appears to be a novel concept.Furthermore, in view of the example from Kang et al, 1991 supra, it issurprising that antigen binding combinations can be produced bycombining the chains from a mouse antibody with repertoires of chainsfrom unimmunised human sources (as described in Example 1 below).

There are a wide range of formats for use of methods according to thepresent invention. For example:

(1) the use of Fv, single chain Fv and Fab fragments is favoured forexpression in bacteria or display on filamentous phage (for Fabfragments see Examples 1 and 2; for scFv see Examples 3 and 4). Mostpreferably, the antibody fragments are displayed on filamentous phage,and the phages selected directly to binding to antigen. The antibodiesselected by these procedures may be used directly, or fused with furthercoding sequences, for example with human constant regions to generateantibody molecules with human effector functions. The variable domainscould also be fused to sequences encoding an enzyme or a toxin moleculeto allow antibody directed targeting of drugs or toxins to particularcell types.

(2) The repertoires of human chains may be readily provided from any ofseveral possible repertoires of V-genes, for example from the rearrangedV-genes of unimmunised humans (Marks et al, 1991, J. Mol. Biol., 222,pp581-597; and Example 1), or from immunised humans, or from syntheticV-gene repertoires. For instance, libraries of human germ line heavy andlight chain V-genes may be generated with synthetic CDR3 regionsincorporating human J regions and antibody specificities isolated(Hoogenboom and Winter, 1992, J. Mol Biol., 227, pp381-388).Furthermore, it may be possible to use repertoires of human V-genes (forexample made synthetically) which are closely related in sequence(perhaps the most homologous) to each of the chains of a mouse antibody.(See Example 5).

(3) The heavy and light chains of the original antibody may be providedby their encoding V-genes (isolated for example by PCR, Orlandi et al.,1989) (Examples 1, 2 and 3) However, it is also possible to start with arepertoire of mouse chains, preferably raised by immunisation (Example4). For example, a repertoire of mouse heavy chains could be combinedwith mouse light chains and selected for binding (as in Clackson et al.,1991 supra): the selected repertoires of paired chains could then berecombined with repertoires of complementary human chains to force newpairings. Alternatively, the repertoire of mouse chains from the mRNA ofan immunised mouse could simply be combined with a repertoire ofcomplementary human chains.

(4) It is possible to use entire heavy and light chain variable domains,as in "chain shuffling" methods or component parts of the chains, forexample as in Marks et al, 1992 supra, by retaining the light chain andCDR3 of the heavy chain of an antibody (and shuffling this sequence witha repertoire of chains comprising the remainder of the heavy chainvariable domain CDRs 1 and 2). One embodiment of the present invention,illustrated by examples 2 and 3, involves retaining at least one regionfrom the original parent non-human antibody, which region contactsantigen, when shuffling with human chains, or component parts of humanantibody antigen-binding site. This region may be a CDR, in which casethe procedure is "CDR-imprinted selection".

Indeed, in examples 2 and 3, the CDR3 of the original mouse heavy chainwas retained, and combined with a repertoire of human heavy chainvariable domains by PCR amplification of a repertoire of human heavychain variable domains with a primer incorporating the mouse CDR3. Theretention of mouse VH-CDR3 may be particularly advantageous in that CDR3of the heavy chain is often most important for antigen binding.

This principle could be extended to a mouse CDR3 repertoire byamplifying the rearranged mouse VH genes with human JH primers (forwardprimers) and human VH framework 3 primers (backward primers). Theseprimers would have to be designed with homology to both mouse and humanV-genes. The amplified DNA repertoire of mouse CDR3s could then beassembled by PCR with a repertoire of human heavy chain genes.

(5) The chain shuffling could be performed using two replicons, forexample using a dual combinatorial procedure as described in Hoogenboomet al, 1991 Nucleic Acids Research, 19, pp4133-4137, in which the chainwhich is to be kept constant is cloned in one replicon, for instance aphage, and then combined with a repertoire of chains in anotherreplicon, for instance a plasmid, as illustrated in Example 1.Alternative methods enabling both chains to be cloned on the samereplicon with high efficiency have also been devised. These again relyon cloning heavy and light chain genes on separate replicons, but thistime with the aim of promoting recombination between two vectors so thatboth chains are placed on the same replicon. An example system is basedon the lox P/Cre recombinase system of coliphage P1 (Hoess and Abremski,1990, in "Nucleic acids and Molecular Biology", Eckstein and Lilley,eds. Vol 4, pp99-109, Springer-Verlag, Berlin, Heidelberg). For the dualcombinatorial procedure the antibody may be expressed as a Fv fragmentor as a Fab fragment (as in Example 1). In the case of a Fab fragment,the VH1CH1 portion of the heavy chain is expressed on one replicon, eg.phage, and the light chain on the other, eg. plasmid. The V domains ofthe Fab fragment to be used in the shuffling procedures as a source ofnew specificities may optionally be fused to cloned human domains eg.Cγ1 and Ck domains. (The V-domains can also be fused directly to theCH1, Ck or Cλ domains by amplifying directly from the human antibodymRNA repertoire.)

Alternatively, the chain shuffling can be performed in a single repliconusing a PCR assembly process as in Clackson et al, 1991, supra, or bysequential cloning of libraries of restriction fragments at appropriatesites which have been incorporated into the original constructs, forinstance into the linker region of a single chain Fv.

The present invention encompasses the replacement of both chains by twoshuffle steps and in one embodiment to the development of a humanantibody with similar or improved characteristics to a non-human, eg.rodent, antibody. In this embodiment, the parent eg rodent antibodywould be made into a "phage antibody". A phage antibody (pAb) is definedas a bacteriophage virus particle which displays at its surface anantibody, an antibody derivative or fragment, such as Fab, Fv, scFv etc,or a domain homologous to an immunoglobulin domain: see WO 92/01047. Onechain would be replaced with a repertoire of chains derived from a humanimmunoglobulin repertoire, for example from an adult lymphocytepopulation or from fetal or artificially derived sources. This mixedpopulation of phage can be selected by binding to antigen to derive apopulation of phage antibodies that bind to the original antigen but nowconsists of one (eg) rodent and one human antibody chain. The remainingrodent chain can be replaced by a repertoire of similar chains derivedfrom a human repertoire, either the same or a different source as wasused in the initial shuffle. After selection a population of antibodiesis obtained which still binds antigen, a population consisting ofantibodies containing two human antibody chains. Thus the originalrodent antibody has been converted into a human antibody that binds thesame antigen.

The generation of a large number of humanised antibodies by methodsaccording to the present invention may have advantages beyond allowingselection of antibodies of desired affinity and specificity. Whenhumanised antibodies are used in humans in therapy it is possible thatthere may be an anti-idiotype response. The antibodies isolated may besurveyed for anti-idiotype response when administered in vivo and thosewith the lowest response selected. Alternatively, one antibody may beadministered therapeutically until an anti-idiotype response is detectedat which time the antibody used is switched to a second, equallyeffective antibody with a different idiotype.

Humanised antibodies which may be obtained using the present inventionare likely to be better than conventional CDR-grafted humanisedantibodies, in the sense that they will be less likely to invoke ananti-idiotypic response.

The antibodies selected by the procedures of the present invention maybe used directly in the single chain Fv or Fab format. Alternatively,the variable domains may be fused with further coding sequences, forexample with human constant regions to generate antibody molecules with,for example, human effector functions. The variable domains could befused to sequences encoding an enzyme or a toxin molecule to allowantibody directed targeting of drugs or toxins to particular cell types(although if these molecules are not of human origin some of theadvantages of humanising are lost).

Although the isolation of antibodies by selection for binding to antigenmay be performed using phage display at each stage, where chainshuffling is performed by PCR assembly or insertion of restrictionfragments, particularly when the numbers have been reduced theantibodies may be screened for antigen binding using a filter assay (A.Skerra et al 1991 Anal Biochem 196 151-155; M. L. Dreher et al 1991 J.Immunol Methods 139 197-205; PCT/GB90/01476). These procedures will beeffective with a relatively small number of clones compared with thenumber which can be handled using phage display.

Aspects and embodiments of the present invention will now be explainedfurther by means of the following examples. These are intended toillustrate the invention without limitation. In addition to thedocuments mentioned throughout the text, attention is drawn to WO92/01047 wherein many of the materials and methods used here weredescribed. The disclosure of WO 92/01047 is herein incorporated byreference. Readers of the present document are urged to consult WO92/01047 for further details and explanation of functional display ofantibodies and antibody fragments, including antibody polypeptidedimers, on the surface of rgdps.

Throughout the text there are references to figures, wherein:

FIG. 1 shows the principle of Epitope Imprinted Selection.

FIG. 2 shows the polylinker region of vector pUC19-pelB-myc. (SEQ.ID NO:126)

FIG. 3 shows results of ELISA of phage displaying Fab fragments, withone chain displayed on phage as a g3p fusion (indicated by fd- . . . ),and the other chain provided as a secreted, soluble chain partner.

FIG. 4 shows a specificity ELISA of the original Mab32 antibody and arange of mouse (scFv-Mab32) and human (other scFv) antibodies.

FIG. 5 shows nucleotide and deduced amino-acid sequences of the V-genesof Mab32. The CDR-regions are in bold. (Heavy chain nucleotides SEQ IDNO:2; Light chain amino acids SEQ ID NO:3.; Light chain nucleotides SEQID NO:4).

FIG. 6 shows the deduced protein sequences of V_(H) and V_(L) antibodygenes of antibody fragments binding to human TNF.

(Light chains: Mab32 SEQ ID NO:110; Vλ1-1-1 SEQ ID NO:111; VλAZ SEQ IDNO:112; VλC4 SEQ ID NO:113; VλD1SEQ ID NO:114. Heavy chains: Mab32 SEQID NO:115; DP-51 SEQ ID NO:116; VHP1SEQ ID NO:117; VHP2 SEQ ID NO:133;VHP3 SEQ ID NO:134; DP-46 SEQ ID NO:118; VHLM2 SEQ ID NO:135; VHLM8 SEQID NO:141).

FIG. 7 shows the results of a competition ELISA between FabMab32 andsingle chain Fv fragments.

FIGS. 8(i) and 8(ii) show an example of a scheme for humanising a mouseantibody using CDR imprinted selection using a dual combinatorialmethod.

FIG. 9 shows an example of a scheme for humanising a mouse antibodyusing CDR imprinted selection using combinatorial libraries in a singlereplicon, single chain Fv format.

FIG. 10 shows relative ELISA signals obtained with scFv fragmentsselected by CDR imprinted selection compared to the original F58 singlechain Fv fragment.

FIG. 11 shows the deduced amino acid sequences of the selected heavychains Graft/27, Graft/2, Graft/H3 and Graft/H9 in comparison to theoriginal F58 heavy chain and to the most closely related human germ lineVH genes. (VH6:F58 SEQ ID NO:127; DP-74 SEQ ID NO:128; Graft/27 SEQ IDNO:136; Graft/2 SEQ ID NO:137; GRAFT/H3 SEQ ID NO:138; GRAFT/H9 SEQ IDNO:139. VH1: F58 SEQ ID NO:129, DP-2 SEQ ID NO:130; Graft/20 SEQ IDNO:140).

FIG. 12 shows the vector pCANTAB3-myc and the DNA and amino acidsequences in the region of the cloning site. (SEQ ID NO:131).

FIG. 13 shows the vector pCANTAB5-myc and the DNA and amino acidsequence in the region of the cloning site. (SEQ ID NO:132).

FIG. 14 shows the assembly of rearranged VH genes.

Table 1 lists oligonucleotide primers and peptides mentioned in thetext. The attention of readers of this document is directed to table 1for sequence information on these oligonucleotides and peptides,complete with the SEQ ID NO of sequences in the listing.

                  TABLE 1                                                         ______________________________________                                        Oligonucleotides Used                                                         PRIMER              SEQUENCE                                                  ______________________________________                                        SEQ ID MOJK1FORNX       5'-CCG TTT GAT TTC                                    NO: 5                   CAG CTT GGT GCC-3'                                    SEQ ID MVKBASFI         5'-CAT GAC CAC GCG                                    NO: 6                   GCC CAG CCG GCC                                                               ATG GCC GAC ATT                                                               GAG CTC ACC CAG                                                               TCT CCA-3'                                            SEQ ID MOVK-HUCK-BACK   5'-GGC ACC AAG CTG                                    NO: 7                   GAA ATC AAA CGG                                                               ACT GTG GCT GCA                                                               CCA TCT GTC TTC-3'                                    SEQ ID HUCKNOT16NOMYC   5'-GAG TCA TTC TCG                                    NO: 8                   ACT TGC GGC CGC                                                               TTA TTA ACA CTC                                                               TCC CCT GTT GAA                                                               GCT CTT-3'                                            SEQ ID VH1FOR-2         5'-TGA GGA GAC GGT                                    NO: 9                   GAC CGT GGT CCC                                                               TTG GCC CC-3'                                         SEQ ID Mo-VH-Hu-CH1     5'-GGG ACC ACG GTC                                    NO: 10                  ACC GTC TCC TCA                                                               GGA AGT GCA TCC                                                               GCC CCA ACC CTT                                                               TTC-3'                                                SEQ ID HCM1FONO         5'-CCA CGA TTC TGC                                    NO: 11                  GGC CGC CAC TGG                                                               AAG AGG CAC GTT                                                               CTT TTC TTT-3'                                        SEQ ID HUCKCYSNOT       5'-GAG TCA TTC TCG                                    NO: 12                  ACT TGC GGC CGC                                                               ACA CTC TCC CCT                                                               GTT GAA GCT CTT-3'                                    SEQ ID HUCLCYSNOT       5'-GAG TCA TTC TCG                                    NO: 12                  ACT TGC GGC CGC                                                               TGA ACA TTC TGT                                                               AGG GGC CAC TGT                                                               CTT-3'                                                SEQ ID MVKBAAPA         5'-CAC AGT GCA CTC                                    NO: 14                  GAC ATT GAG CTC                                                               ACC CAG TCT CCA-3'                                    SEQ ID VH1BACKAPA       5'-CAT GAC CAC AGT                                    NO: 107                 GCA CAG GTS MAR                                                               CTG CAG SAG TCW                                                               GG-3'                                                 SEQ ID HCM1FONO         5'-CCA CGA TTC TGC                                    NO: 15                  GGC CGC CAC TGG                                                               AAG AGG CAC GTT                                                               CTT TTC TTT-3'                                        SEQ ID HCM1FO           5'-TGG AAG AGG CAC                                    NO: 16                  GTT CTT TTC TTT-3'                                    SEQ ID HUCLCYS          5'-TGA ACA TTC TGT                                    NO: 17                  AGG GGC CAC TGT                                                               CTT-3'                                                SEQ ID HUCKCYS          5'-ACA CTC TCC CCT                                    NO: 18                  GTT GAA GCT CTT-3'                                    SEQ ID fd-PCR-BACK      5'-GCG ATG GTT GTT                                    NO: 19                  GTC ATT GTC GGC-3'                                    SEQ ID fd-SEQ1          5'-GAA TTT TCT GTA                                    NO: 20                  TGA GG-3'                                             SEQ ID HULAMBDASEQ      5'-GTG TGG CCT TGT                                    NO: 21                  TGG CTT G-3'                                          SEQ ID pHEN-SEQ         5'-CTA TGC GGC CCC                                    NO: 22                  ATT CA-3'                                             SEQ ID LINKSEQ          5'-CGA TCC GCC ACC                                    NO: 23                  GCC AGA G-3'                                          SEQ ID Hu-MCH1FORSEQ2   5'-AGG AAG TCC TGT                                    NO: 24                  GCG AGG CAG-3'                                        SEQ ID HuVL1BACKSFI     5'-GTC CTC GCA ACT                                    NO: 25                  CGC GCC CAG CCG                                                               GCC ATG GCC CAG                                                               TCT GTG TTG ACG                                                               CAG CCG CC-3'                                         SEQ ID HuCL1FORAMBNOT   5'-CCA CGA TTC TGC                                    NO: 26                  GGC CGC CTA TGA                                                               ACA TTC TGT AGG                                                               GGT CAC TGT-3'                                        SEQ ID VH1BACKSFI15     5'-CAT GCC ATG ACT                                    NO: 108                 CGC GGC CCA GCC                                                               GGC CAT GGC CSA                                                               GGT SMA RCT GCA                                                               GSA GTC (A/T)GG                                       SEQ ID HuJH4FORASSXHoI  5'-GCCTGAACCGCC                                       NO: 27                  TCCACCTCTCGAGAA                                                               CGGTGACCAGGG-3'                                       SEQ ID HuVBLBACK-XhoI   5'-CTGGTCACCGTC                                       NO: 28                  TCGAGAGGTGGA                                                                  GGC-3'                                                SEQ ID HuJH4FORSa1I     5'-GGAGGATGCACT                                       NO: 29                  TGTCGACACGGT                                                                  GACCAG-3'                                             Human VH Back Primers                                                         SEQ ID HuVH1aBACK       5'-CAG GTG CAG CTG                                    NO: 30                  GTG CAG TCT GG-3'                                     SEQ ID HuVH2aBACK       5'-CAG GTC AAC TTA                                    NO: 31                  AGG GAG TCT GG-3'                                     SEQ ID HuVH3aBACK       5'-GAG GTG CAG CTG                                    NO: 32                  GTG GAG TCT GG-3'                                     SEQ ID HuVH4aBACK       5'-CAG GTG CAG CTG                                    NO: 33                  CAG GAG TCG GG-3'                                     SEQ ID HuVH5aBACK       5'-GAG GTG CAG CTG                                    NO: 34                  TTG CAG TCT GC-3'                                     SEQ ID HuVH6aBACK       5'-CAG TGA CAG CTG                                    NO: 35                  CAG CAG TCA GG-3'                                     Human IgM Constant Region Primer                                              SEQ ID HuIgMFOR         5'-TGG AAG AGG CAC                                    NO: 36                  GTT CTT TTC TTT-3'                                    Human κ Constant Region Primer                                          SEQ ID HUCκFOR    5'-AGA CTC TCC CCT                                    NO: 37                  GTT GAA GCT CTT-3'                                    Human λ Constant Region Primer                                         SEQ ID HUCλFOR   5'-TGA AGA TTC TGT                                    NO: 38                  AGG GGC CAC TGT                                                               CTT-3'                                                Human JH Forward Primers                                                      SEQ ID HuJH1-2FOR       5'-TGA GGA GAC GGT                                    NO: 39                  GAC CAG GGT GCC-3'                                    SEQ ID HuJH3FOR         5'-TGA AGA GAC GGT                                    NO: 40                  GAC CAT TGT CCC-3'                                    SEQ ID HuJH4-5FOR       5'-TGA GGA GAC GGT                                    NO: 41                  GAC CAG GGT TCC-3'                                    SEQ ID HuJH6FOR         5'-TGA GGA GAC GGT                                    NO: 42                  GAC CGT GGT CCC-3'                                    VH BACK PRIMERS WITH APA LI SITES                                             SEQ ID HUVH1BAAPA       5'-CAT GAC CAC AGT                                    NO: 43                  GCA CAG GTG CAG                                                               CTG GTG CAG TCT                                                               GG-3'                                                 SEQ ID HUVH2BAAPA       5'-CAT GAC CAC AGT                                    NO: 44                  GCA CAG GTC AAC                                                               TTA AGG GAG TCT                                                               GG-3'                                                 SEQ ID HUVH3BAAPA       5'-CAT GAC CAC AGT                                    NO: 45                  GCA CAG GTG CAG                                                               CTG GTG GAG TCT                                                               GG-3'                                                 SEQ ID HUVH4BAAPA       5'-CAT GAC CAC AGT                                    NO: 46                  GCA CAG GTG CAG                                                               CTG CAG GAG TCG                                                               GG-3'                                                 SEQ ID HUVH5BAAPA       5'-CAT GAC CAC AGT                                    NO: 47                  GCA CAG GTG CAG                                                               CTG TTG CAG TCT                                                               GC-3'                                                 SEQ ID HUVH6BAAPA       5'-CAT GAC CAC AGT                                    NO: 48                  GCA CAG GTA CAG                                                               CTG CAG CAG TCA                                                               GG-3'                                                 JHSAL primers                                                                 SEQ ID HUJH1-2FORSAL    5'-GAG TCA TTC TCG                                    NO: 49                  TGT CGA CAC GGT                                                               GAC CAG GGT GCC-3'                                    SEQ ID HUJH3FORSAL      5'-GAG TCA TTC TCG                                    NO: 50                  TGT CGA CAC GGT                                                               GAC CAT TGT CCC-3'                                    SEQ ID HUJH4-5FORSAL    5'-GAG TCA TTC TCG                                    NO: 51                  TGT CGA CAC GGT                                                               GAC CAG GGT TCC-3'                                    SEQ ID HUJH6FORSAL      5'-GAG TCA TTC TCG                                    NO: 52                  TGT CGA CAC GGT                                                               GAC CGT GGT CCC-3'                                    LAMBDA BACK PRIMERS WITH SFI SITES                                            SEQ ID HUVLIBASFI:      5'-GTC CTC GCA ACT                                    NO: 53                  GCG GCC CAG CCG                                                               GCC ATG GCC CAG                                                               TCT GTG TTG ACG                                                               CAG CCG CC-3'                                         SEQ ID HUVL2BASFI:      5'-GTC CTC GCA ACT                                    NO: 54                  GCG GCC CAG CCG                                                               GCC ATG GCC CAG                                                               TCT GCC CTG ACT                                                               CAG CCT GC-3'                                         SEQ ID HUVL3aBASFI:     5'-GTC CTC GCA ACT                                    NO: 55                  GCG GCC CAG CCG                                                               GCC ATG GCC TCC                                                               TAT GTG CTG ACT                                                               CAG CCA CC-3'                                         SEQ ID HUVL3bBASFI:     5'-GTC CTC GCA ACT                                    NO: 56                  GCG GCC CAG CCG                                                               GCC ATG GCC TCT                                                               TCT GAG CTG ACT                                                               CAG GAC CC-3'                                         SEQ ID HUVL4BASFI:      5'-GTC CTC GCA ACT                                    NO: 57                  GCG GCC CAG CCG                                                               GCC ATG GCC CAG                                                               GTT ATA CTG ACT                                                               CAA CCG CC-3'                                         SEQ ID HUVL5BASFI:      5'-GTC CTC GCA ACT                                    NO: 58                  GCG GCC CAG CCG                                                               GCC ATG GCC CAG                                                               GCT GTG CTC ACT                                                               CAG CCG TC-3'                                         SEQ ID HUVL6BASFI:      5'-GTC CTC GCA ACT                                    NO: 59                  GCG GCC CAG CCG                                                               GCC ATG GCC AAT                                                               TTT ATG CTG ACT                                                               CAG CCC CA-3'                                         KAPPA BACK PRIMERS WITH SFI SITES                                             SEQ ID HUVK1BASFI:      5'-GTC CTC GCA ACT                                    NO: 60                  GCG GCC CAG CCG                                                               GCC ATG GCC GAC                                                               ATC CAG ATG ACC                                                               CAG TCT CC-3'                                         SEQ ID HUVK2BASFI:      5'-GTC CTC GCA ACT                                    NO: 61                  GCG GCC CAG CCG                                                               GCC ATG GCC GAT                                                               GTT GTG ATG ACT                                                               CAG TCT CC-3'                                         SEQ ID HUVK3BASFI:      5'-GTC CTC GCA ACT                                    NO: 62                  GCG GCC CAG CCG                                                               GCC ATG GCC GAA                                                               ATT GTG TTG ACG                                                               CAG TCT CC-3'                                         SEQ ID HUVK4BASFI:      5'-GTC CTC GCA ACT                                    NO: 63                  GCG GCC CAG CCG                                                               GCC ATG GCC GAC                                                               ATC GTG ATG ACC                                                               CAG TCT CC-3'                                         SEQ ID HUVK5BASFI:      5'-GTC CTC GCA ACT                                    NO: 64                  GCG GCC CAG CCG                                                               GCC ATG GCC GGA                                                               ACG ACA CTC ACG                                                               CAG TCT CC-3'                                         SEQ ID HUVK6BASFI:      5'-GTC CTC GCA ACT                                    NO: 65                  GCG GCC CAG CCG                                                               GCC ATG GCC GAA                                                               ATT GTG CTG ACT                                                               CAG TCT CC-3'                                         SEQ ID HUCLFORSERASCNOT 5'-GAG TCA TTC TCG                                    NO: 66                  ACT TGC GGC CGC                                                               CTG CTA TTA TCG                                                               GGC GCG CCT TTA                                                               TTA TGA AGA TTC                                                               TGT AGG GGC CAC                                                               TGT CTT-3'                                            SEQ ID HUCKFORSERASCNOT 5'-GAG TCA TTC TCG                                    NO: 67                  ACT TGC GGC CGC                                                               CTG CTA TTA TCG                                                               GGC GCG CCT TTA                                                               TTA AGA CTC TCC                                                               CCT GTT GAA GCT                                                               CTT-3'                                                SEQ ID HUCKFORSER       5'-AGA CTC TCC CCT                                    NO: 68                  TTT GAA GCT CTT-3'                                    SEQ ID HUCLFORSER       5'-TGA AGA TTC TGT                                    NO: 69                  AGG GGC CAC TGT                                                               CTT-3'                                                SEQ ID HUCKFORSERNOT    5'-GAG TCA TTC TCG                                    NO: 70                  ACT TGC GGC GGC                                                               AGA CTC TCC CCT                                                               GTT GAA GCT CTT-3'                                    SEQ ID HUCLFORSERNOT    5'-GAG TCA TTC TCG                                    NO: 71                  ACT TGC GGC CGC                                                               TGA AGA TTC TGT                                                               AGG GGC CAC TGT                                                               CTT-3'                                                Human Vκ Back Primers                                                   SEQ ID HuVk1aBACK       5'-GAC ATC CAG ATG                                    NO: 72                  ACC CAG TCT CC-3'                                     SEQ ID HuVk2aBACK       5'-GAT GTT GTG ATG                                    NO: 73                  ACT CAG TCT CC-3'                                     SEQ ID HuVk3aBACK       5'-GAA ATT GTG TTG                                    NO: 74                  ACG CAG TCT CC-3'                                     SEQ ID HuVK4aBACK       5'-GAC ATC GTG ATG                                    NO: 75                  ACC CAG TCT CC-3'                                     SEQ ID HuVk5aBACK       5'-GAA ACG ACA CTC                                    NO: 76                  ACG CAG TCT CC-3'                                     SEQ ID HuVk6aBACK       5'-GAA ATT GTG CTG                                    NO: 77                  ACT CAG TCT CC-3'                                     Human λ Back Primers                                                   SEQ ID Huλ1BACK  5'-CAG TCT GTG TTG                                    NO: 78                  ACG CAG CCG CC-3'                                     SEQ ID HuλBACK   5'-CAG TCT GCC CTG                                    NO: 79                  ACT CAG CCT GC-3'                                     SEQ ID Huλ3aBACK 5'-TCC TAT GTG CTG                                    NO: 80                  ACT CAG CCA CC-3'                                     SEQ ID Huλ3bBACK 5'-TCT TCT GAG CTG                                    NO: 81                  ACT CAG GAC CC-3'                                     SEQ ID Huλ4BACK  5'-CAC GTT ATA CTG                                    NO: 82                  ACT CAA CCG CC-3'                                     SEQ ID Huλ5BACK  5'-CAG GCT GTG CTC                                    NO: 83                  ACT CAG CCG TC-3'                                     SEQ ID Huλ6BACK  5'-AAT TTT ATG CTG                                    NO: 84                  ACT CAG CCC CA-3'                                     SEQ ID G3LASCGTGBACK    5'-GTC CTC GCA ACT                                    NO: 85                  GGC GCG CCA CAA                                                               TTT CAC AGT AAG                                                               GAG GTT TAA CTT                                                               GTG AAA AAA TTA                                                               TTA TTC GCA ATT-3'                                    SEQ ID F58GRAFTJH4SAL   5'-GGA TGC ACT TGT                                    NO: 86                  CGA CAC GGT GAC                                                               CAG GGT ACC TTG                                                               GCC CCA GTA GTC                                                               AAA GTA GTA GTC                                                               CTC TTC GTA ATC                                                               ATA GTA GAT CAG                                                               GTC ACA GTA ATA                                                               CAC GGC CGT GTC-3'                                    SEQ ID F58GRAFTSAL      5'-GGA TGC ACT TGT                                    NO: 87                  CGA CAC GGT GAC                                                               CAG GGT ACC TTG                                                               GCC CCA GTA GTC                                                               AAA GTA GTA GTC                                                               CTC TTC GTA ATC                                                               ATA GTA GAT CAG                                                               GTC ACA GTA ATA                                                               CAC GGC CGT GTC-3'                                    LAMBDA BACK PRIMERS WITH APA LI SITES                                         SEQ ID HUVL1BAAPA:      5'-TGA GCA CAC AGT                                    NO: 88                  GCA CTC CAG TCT                                                               GTG TTG ACG CAG                                                               CCG CC-3'                                             SEQ ID HUVL2BAAPA:      5'-TGA GCA CAC AGT                                    NO: 89                  GCA CTC CAG TCT                                                               GCC CTG ACT CAG                                                               CCT GC-3'                                             SEQ ID HUVL3aBAAPA:     5'-TGA GCA CAC AGT                                    NO: 90                  GCA CTC TCC TAT                                                               GTG CTG ACT CAG                                                               CCA CC-3'                                             SEQ ID HUVL3bBAAPA:     5'-TGA GCA CAC AGT                                    NO: 91                  GCA CTC TCT TCT                                                               GAG CTG ACT CAG                                                               GAC CC-3'                                             SEQ ID HUVL4BAAPA:      5'-TGA GCA CAC AGT                                    NO: 92                  GCA CTC CAG GTT                                                               ATA CTG ACT CAA                                                               CCG CC-3'                                             SEQ ID HUVL5BAAPA:      5'-TGA GCA CAC AGT                                    NO: 93                  GCA CTC CAG GCT                                                               GTG CTC ACT CAG                                                               CCG TC-3'                                             SEQ ID HUVL6BAAPA:      5'-TGA GCA CAC AGT                                    NO: 94                  GCA CTC AAT TTT                                                               ATG CTG ACT CAG                                                               CCC CA-3'                                             KAPPA BACK PRIMERS WITH APA LI SITES                                          SEQ ID HUVK1BAAPA:      5'-TGA GCA CAC AGT                                    NO: 95                  GCA CTC GAC ATC                                                               CAG ATG ACC CAG                                                               TCT CC-3'                                             SEQ ID HUVK2BAAPA:      5'-TGA GCA CAC AGT                                    NO: 96                  GCA CTC GAT GTT                                                               GTG ATG ACT CAG                                                               TCT CC-3'                                             SEQ ID HUVK3BAAPA:      5'-TGA GCA CAC AGT                                    NO: 97                  GCA CTC GAA ATT                                                               GTG TTG ACG CAG                                                               TCT CC-3'                                             SEQ ID HUVK4BAAPA:      5'-TGA GCA CAC AGT                                    NO: 98                  GCA CTC GAC ATC                                                               GTG ATG ACC CAG                                                               TCT CC-3'                                             SEQ ID HUVK5BAAPA:      5'-TGA GCA CAC AGT                                    NO: 99                  GCA CTC GAA ACG                                                               ACA CTC ACG CAG                                                               TCT CC-3'                                             SEQ ID HUVK6BAAPA:      5'-TGA GCA CAC AGT                                    NO: 100                 GCA CTC GAA ATT                                                               GTG CTG ACT CAG                                                               TCT CC-3'                                             Human VH Back Primers                                                         SEQ ID HuVH1aBACKSfi    5'-GTC CTC GCA ACT                                    NO: 101                 GCG GCC CAG CCG                                                               GCC ATG GCC CAG                                                               GTG CAG CTG GTG                                                               CAG TCT GG-3'                                         SEQ ID HuVH2aBACKSfi    5'-GTC CTC GCA ACT                                    NO: 102                 GCG GCC CAG CCG                                                               GCC ATG GCC CAG                                                               GTC AAC TTA AGG                                                               GAG TCT GG-3'                                         SEQ ID HuVH3aBACKSfi    5'-GTC CTC GCA ACT                                    NO: 103                 GCG GCC CAG CCG                                                               GCC ATG GCC CAG                                                               GTG CAG CTG GTG                                                               GAG TCT GG-3'                                         SEQ ID HuVH4aBACKSfi    5'-GTC CTC GCA ACT                                    NO: 104                 GCG GCC CAG CCG                                                               GCC ATG GCC CAG                                                               GTG CAG CTG CAG                                                               GAG TCG GG-3'                                         SEQ ID HuVH5aBACKSfi    5'-GTC CTC GCA ACT                                    NO: 105                 GCG GCC CAG CCG                                                               GCC ATG GCC CAG                                                               GTG CAG CTG TTG                                                               CAG TCT GC-3'                                         SEQ ID HuVH6aBACKSfi    5'-GTC CTC GCA ACT                                    NO: 106                 GCG GCC CAG CCG                                                               GCC ATG GCC CAG                                                               GTA CAG CTG CAG                                                               CAG TCA GG-3'                                         SEQ ID SYNLIB1          5'-GCC TCC ACC TCT                                    NO: 109                 CGA GAC GGT GAC                                                               CAG GGT ACC TTG                                                               GCC CCA ATA GTC                                                               AAA ((A/C)NN)5 TCT                                                            TGC ACA GTA ATA                                                               CAC GGC CGT GTC-3'                                    ______________________________________                                    

The examples illustrate embodiments of processes according to thepresent invention as follows:

In the work described in example 1, a mouse antibody directed against anepitope of TNF was cloned as a Fab fragment for display on phage, and bycombining the heavy chain with repertoires of human light chains (orindeed the light chain with repertoires of human heavy chains), it waspossible to select phage bearing Fab fragments with one mouse and onehuman chain. These antibody fragments bound to the same epitope of TNFas the original mouse antibody. The new human chain (heavy or light) wasthen combined with a repertoire of human partner chains to create anentirely human antibody Fab fragment which binds to the same epitope ofTNF.

Example 2 illustrates CDR imprinted selection in a dual combinatorialformat. An anti-HIV gp120 antibody was humanised by epitope imprintedselection with retention of the original mouse heavy chain CDR3. The VHand VL domains of the mouse antibody were cloned as fusions with mouseCH1 and Ck chains. The resulting heavy chain VHCH1 fragment wasdisplayed on phage in combination with a repertoire of human lightchains expressed as a g3p fusion from phage vector. Human light chainsdisplayed on phage were selected and the selected chains displayed inphage in combination with a library of human naive heavy chain VHCH1fragments amplified so as to comprise the CDR3 of the original mouseheavy chain, expressed as g3p fusions. Selected phage were obtaineddisplaying antibody polypeptide dimers which retain the originalspecificity against gp120 but are now of fully human origin apart fromretaing the mouse heavy chain CDR3.

Example 3 illustrates CDR imprinted selection in a single replicon,single chain Fv format. An anti-HIV gp120 antibody was humanised byepitope imprinted selection with retention of the original mouse heavychain CDR3. A single chain Fv library was displayed on phage, thefragments comprising the original mouse light chain and CDR3 of theheavy chain but with the remainder of the light being of human origin,derived from a library of human naive heavy chains. The heavy chainvariable domain of selected phage was then combined with a repertoire ofhuman light chain variable domains to generate a scFv library on phage.Phage selected from this library displayed scFv fragments in which thevariable domains were of fully human origin except for the retention ofCDR3 of the mouse heavy chain.

Example 4 illustrates the isolation of human antibodies derived from theimmune repertoire of an immunised mouse. A repertoire of mouse heavy andlight chain V genes derived from a mouse immunised with TNF aredisplayed as single chain Fv fragments and phage selected for binding toTNF. VH genes derived from this selected mouse population are thencombined with VL genes derived from a human naive library as singlechain Fv fragments and phage selected. Selected semi-humanised antibodypolypeptide dimers are then fully humanised by pairing the human VLswith human VHs derived from a human naive library. Fully humanisedantibody polypeptide dimers specific for TNF may then be selected fromthe resulting library.

Example 5 illustrates creation of a synthetic library.

EXAMPLE 1

Humanisation of a murine antibody: Isolation of human antibodiesdirected against a specific epitope on tumour necrosis factor alpha(TNF-alpha) using epitope imprinted selection

We chose a murine monoclonal antibody directed against human TNF as ourtarget antibody for humanisation by double chain shuffling. The antibodyMab32 (class IgG2b, k) (D. Rathjen et al., 1991Mol. Immunol. 28, p79; D.Rathjen et al, 1992 Brit. J. Cancer 65, pp852-856; Australian PatentApplication 7,576; EP 486,526) does not inhibit the cytolytic effect ofTNF (presumably because it does not inhibit the binding of TNF to itsreceptors), but it enhances the in vivo anti-cancer and anti-viraleffects of the cytokine. The antibody binding site is localised in aregion of the TNF-trimer, which encompasses the first 18 residues ofTNF. This example demonstrates that epitope imprinted selection can beused to generate human antibodies specifically recognising this sameepitope on human TNF-alpha.

Experimental and Results

General methods used are given at the end of this section.

1 Cloning and display of the V genes of Mab 32 on phase

Cloning of the V-genes of Mab32:

The genes of the mouse Mab32 antibody (IgG2b, Kappa) were rescued by PCRessentially as described (Clackson et al., 1991, supra, Clackson et alin "PCR: a practical approach, eds Mr Phenox et al, IRL Press, Oxford pp187-214) using the primers VH1BACk and VH1FOR2 for the VH gene andVK2BACK and VK4FOR for the VL gene and the polymerase chain reaction(PCR, R. K. Saiki et al., 1985, Science 230, p1350). The mouse VH and Vkgenes were assembled for expression as scFv fragments by PCR assembly(Clackson et al., supra) amplified with VH1BACKSfi and VKFOR4NOT andligated into phagemid pHEN1 (H. R. Hoogenboom et. al., 1991 Nucl. Acids.Res. 19, pp4133-4137) as a SfiI-NotI cut restriction fragment, andelectroporated into E. coli HB2151 cells. Of 96 clones analysed by ELISA(see below), 9 secreted TNF-binding soluble scFv fragments. Sequencingrevealed in all clones a mouse VH of family IIB and a mouse Vk of familyVI (E. A. Kabat et al., 1991 Sequences of Proteins of ImmunologicalInterest, U.S. Public Health Services). Nucleotide mutations which wereprobably introduced by the PCR were detected by comparing the 9sequences, and a clone with `consensus` sequence and binding activity(scFv-Mab32) chosen for further cloning experiments (FIG. 5).

Recloning of the Mab32 V-genes for soluble expression:

The murine V-genes were recloned for soluble expression of heavy (Fd,VHCH1) or light chain, by linking the mouse V-genes to the human CH1 (ofthe mu-isotype) or human Ck gene respectively by splice overlapextension. The mouse Vk gene was amplified from scFv-Mab32 DNA witholigonucleotides MOJK1FORNX (binds in joining region of V-gene; thesequences of all oligonucleotides used in these examples are shown inTable 1) and MVKBASFI (binds in 5' region and adds SfiI restrictionsite); the human Ck was obtained by PCR from a mouse-human chimaericlight chain gene (of NQ10.12.5, described in Hoogenboom et al., 1991,supra), with oligonucleotides MOVK-HUCK-BACK (binds in 5' of human Ckand is partially complementary with mouse Jk 1 region) andHUCKNOT16NOMYC (sits in 3' end of human Ck, retains the terminalcysteine, and tags on a NotI restriction site) as in Clackson et al,1991 using a two fragment assembly. For linkage of the DNA fragments,the two PCR fragments were mixed and amplified with MVKBASFI andHUCKNOT16NOMYC. The chimaeric VkCk gene was subsequently cloned as aSfiI-NotI fragment in pUC19 derivative containing the pelB signalpeptide sequence and appropriate cloning sites for soluble expression ofthe light chain (pUC19-pelB-myc, FIG. 2). Similarly, the mouse VH gene(amplified from scFv-Mab32 with LMB3 and VH1FOR-2) was combined bysplicing by overlap extension PCR with the human u-CH1 domain (amplifiedfrom human IgM-derived cDNA (Marks et al., 1991, supra WO 92/01047) withMo-VH-Hu-CH1 and HCM1FONO, and cloned as SfiI-NotI fragment into apUC19-pelB-myc for soluble expression of a tagged chain.

Display of the Mab32 antibody on phage:

The chimaeric light chain was displayed on phage fd by reamplificationof the mouse/human chimaeric chain with HUCKCYSNOT and MVKBAAPA andcloning into fd-tet-DOG1 as an ApaLI-NotI fragment. Cells harbouring aplasmid with the heavy Fd chain gene were grown in 2xTY containingAMP-GLU (1%) to logarithmic phase (OD600 of 0.5) and infected with a20-fold excess of light-chain displaying phage. After 45 min at 37'C.without shaking and 45 min at 37'C. with shaking in the 2xTY, ampicillin(100 ug/ml), Glucose 1% medium, a sample was diluted into 50-fold volumeof prewarmed (37'C.) 2 x TY, ampicillin (100 ug/ml) and tetracyclin (15ug/ml, grown for 1 hr at 37'C. and then overnight at 30'C. (shaking).Phage particles collected from the supernatant of such culture displayedTNF-binding Fab fragments anchored through the light chain on theirsurface.

Similarly, the reversed configuration was made. The heavy chain VHCH1fragment was cloned into fd-tet-DOG1 (after amplification of the Fdchain gene from the mouse/human chimeric construct with VH1BACKAPA andHCM1FONO), and phage used to infect cells capable of producing solublelight chain. Phage particles collected from the supernatant of suchculture displayed TNF-binding Fab fragments anchored through the heavychain VHCH1 fragment on their surface.

Properties of Mab32 fragments displayed on phage:

The V-genes of the murine antibody Mab32 were cloned by amplifying thehybridoma V-genes, cloning the VH and Vk genes as scFv fragments inphagemid pHEN1 as above. Antibody scFv fragments which bind to TNF wereidentified by ELISA (see below for details). The mouse VH gene wasrecloned in pUC19-pelB-myc (FIG. 2) for soluble expression as a mouse VHlinked to human mu-CH1, while the light chain was recloned with thehuman Ck domain in vector fd-tet-DOG1 as a fusion with g3p. When cellsharbouring the heavy chain construct were infected with the fd-phagecarrying the light chain, phage particles emerged which carried lightchain-g3p associated with the Fd heavy chain. Indeed, binding to TNF andthe 301 peptide was retained, as judged by ELISA with phage displayingthe mouse-human chimaeric Fab fragment (FIG. 3). In the phage ELISA, thebackground signal of phage carrying the light chain only was s lightlyhigher than wild-type fd-tet-DOG1 phage, but always lower than thesignal obtained with Fab-displaying phage. Similarly, TNF binding phagewas made with the heavy chain VHCH1 fragment anchored on phage, and thelight chain provided as a soluble fragment. Hence, Mab32 is functionalin the dual combinatorial format in both display orientations.

2 Chain shuffling by epitope imprinted selection (EIS)

Construction of One chain-Libraries:

Kappa, lambda light chain and Mu-specific cDNA was made from the mRNAprepared from the peripheral blood lymphocytes from two healthy donorsessentially as in Marks et al., 1991, supra. The first-strand cDNAsynthesis was performed with oligonucleotides HCM1FO, HUCLCYS andHUCKCYS for Mu-specific, lambda and kappa libraries respectively. TheVH-CH 1 repertoire was amplified from this cDNA with oligonucleotidesHCM1FO and six family specific VHBACK primers (as in Marks et al., 1991,supra), reamplified with a NotI-tagged forward primer (HCM1FONO) andApaLI tagged VHBACK primers (6 primers HuVH1BAAPA to HuVH6BAAPA: Table1). Similarly, the light chain repertoires were amplified with HUCLCYSor HUCKCYS forward primers and HUVλ1BACK to HuVλ6BACK or HuVk1BACK toHuVk6BACK back primers described in Marks et al., 1991, supra andPCT/GB91/01134 (WO 92/01047). In each case described in this section thelambda and kappa chain variable repertoires were amplified separately.The amplified repertoires were reamplified with ApaLI and NotI taggedversions of these oligonucleotides (13 back primers HuVλ1BAAPA toHuVλ6BAAPA or HuVk1BAAPA to HuVkBAAPA and two forward primers HuCLCYSNOTand HuCKCYSNOT, respectively). All three repertoires were cloned intovector fd-tet-DOG1 as ApaLI-NotI fragments, and electroporated into E.coli MC1061 cells, to obtain libraries of 1.0×10⁷ clones for VλCλ,1.4×10⁷ clones for VkCk, and 5×10⁶ clones for IgM-derived VHCH1. Thepresence of insert was checked and the frequency of inserts in thelibrary found to be higher than 95% in all three cases.

Selecting a human VL using the mouse VH domain as dockinq chain:

When chain shuffling using epitope imprinted selection, there are tworoutes from the mouse to the human antibody, depending on which mouseV-gene is kept constant in the first rounds of selection (see FIG. 1).The influence of the mouse heavy and light chain in binding to antigenand their influence on docking human partner chains onto the originalepitope may vary considerably between antibodies, perhaps depending onwhich domain forms most of the antigen-binding contacts.

FIG. 1 shows the principle of Epitope Imprinted Selection, asexemplified using a mouse antibody to start with. The clear rectanglesrepresent the original mouse antibody VH and VL domains. Shadedrectangles represent human VH and VL domains. The human antibodiesderived shown on the left on the bottom line are produced by a procedureinvolving:

(a) (i) retention of the original mouse VH and shuffling with a libraryof human VL domains followed by selection by binding to antigen.

(a) (ii) shuffling of these selected human VL domains with a library ofhuman VH domains followed by selection by binding to antigen.

The human antibodies derived shown on the right on the bottom line arederived by the alternative pathway involving:

(b) (i) retention of the original mouse VL and shuffling with a libraryof human VH domains followed by selection by binding to antigen.

(b) (ii) shuffling of these selected human VH domains with a library ofhuman VL domains followed by selection by binding to antibody.

The human antibodies in the middle of the bottom line are derived by;

(c) shuffling the selected human VL chains derived in (a) (i) with theselected human VH chains derived in (b) (i) and then selecting bybinding to antigen.

VH chains derived in (b) (ii) can be-combined with VL chains derived in(a) (ii) and vice versa.

The current section describes procedure (a) and subsequent sectionsprocedures (b) and (c). This example describes the procedures in a dualreplicon, dual combinatorial format although the methods are equallyapplicable to a single replicon, single chain Fv format.

In the first chain shuffling experiment, the mouse VH (linked to thehuman CH1 domain), expressed from pUC19-pelB-myc, was paired as Fabfragment with a library of 10⁷ different human VλCλ domains. Phagedisplaying the antibody fragments were subjected to rounds of panning onTNF-coated tubes. By following the titre of the eluted phage, the extentof selection can be monitored. After 4 rounds (with a 100-fold increasein the titre of eluted phage), 24 out of 28 individual clones were foundto be binding to TNF in an ELISA with phage expressing Fab fragments(all with the mouse VH-human CH1). Phage only displaying the selectedhuman VλCλ domains gave a background similar to phage displaying onlythe chimaeric mouseVk-human Ck. Sixteen clones taken after the firstround of selection were found to be negative.

Only three different BstNI fingerprints were found amongst the 24binders, with one pattern dominating (21/24). Light chains VλA2, VλC4and VλD1 were found with frequencies of 21/24, 2/24. and 1/24respectively. Sequencing revealed that all three light chains arederived from the same germline gene, a human Vλ1-1-1. Clone VλC4 has 1,clone VλD1 has 2 and clone VλA2 7 amino-acid residue differences fromthe germline (FIG. 6). However, clone VλA2 uses a framework-1 regionwhich more closely resembles the germline sequence of a related Vλ1,humv1117, and therefore may be the result of a cross-over. The germlinecharacter of the clones is also noted in the CDR3 sequence, with minimalvariation in sequence and no length variation between the three clones.Apparently, only a very limited number of genes with very similarsequences fit the stringent requirements (being compatible with themouse VH and forming an antigen-binding pair). One of the surprises isthat human germline encoded V-genes can supply domains that fit thoseconditions. However, the starting material of this library has proven tobe very diverse in other experiments. For example, from an unimmunisedscFv library made from the same two donors, we derived many different λchains with a range of nucleotide mutations compared to known germlinegenes (Marks et al., 1991, supra). In addition, light chain genesderived from the same donors were used in a chain shuffling experimentfor affinity maturation, and light chain variants (all Vλ1) withnumerous mutations isolated (Marks et al., 1992, supra).

Selecting a human VH using the selected human VL domains as dockingchains:

The 3 selected Vλ genes were recloned in pUC19-pelB-myc for solubleexpression as VλCλ chains. E. coli cells harbouring the three lightchain plasmids were mixed, infected with a phage library of human VHCH1genes, expressed from the fd-tet-DOG1 library described earlier and thelibrary subjected to rounds of panning on TNF-coated Immuno tubes.Clones were picked after 5 rounds, when the titre of eluted phageincreased 100-fold. Fifteen out of 20 clones analysed by BstNIfingerprint of the DNA insert used one of two patterns (withapproximately the same frequency). The 15 clones when combining theirheavy chain VHCH1 fragments with the VλA2 light chain gave strongerphage ELISA signals than when combined with the VλC4 or VλD1 light chain(FIG. 3). Background signals obtained with phage displaying the heavychain VHCH1 fragment only were similar to the signal of the murineVH-human CH1.

Sequencing revealed that the two patterns could be assigned to threeunique human VH sequences (clones VHP1/2/3, with clone VHP1 having aBstNI fingerprint which is nearly identical to that of clone VHP2). Likethe selected light chain genes, the selected heavy chain genes arederived from the same germline VH gene (germline DP-51 from the VH3family, Tomlinson et al., J. Mol. Biol. 227, pp776-798 1992; FIG. 6),with minimal residue differences. FIG. 6 shows the deduced proteinsequences of V_(H) and V_(L) antibody genes of antibody fragmentsbinding to human TNF. The selected human V-genes were aligned to theirclosest germline homologue; identical residues in the selected genes arerepresented by hyphens. Framework 4 of the V_(H) genes has beentruncated at 4th residue. Clone VHP1 most likely is a cross-over betweenDP-51 and a related germline, DP-47. All three selected VH-genes haverelatively short CDR3 loops (8,9 and 10 residues), but share littlehomology in this sequence. Since the VH-CDR3 is naturally the mostdiverse region of all six antibody variable loops, the chance ofselecting clones with a consensus in that region is very low indeed.

Specificity of binding of the selected V-gene pairs:

A specificity ELISA with Mab32 and soluble scFv fragments on a number ofantigens showed that the original Mab32 antibody, its scFv-derivativeand three of the humanised TNF-binders (as scFv-fragments) bindspecifically to TNF (FIG. 4). No significant binding was obtained toELISA plates coated with keyhole limpet haemocyanin, ovalbumin,cytochrome c, bovine serum albumin, human thyroglobulin, or2-phenyloxazol-5-one-BSA or to plastic only. Proof that the humanisedantibodies bind to the same epitope came first from phage ELISA withpeptide.301-coated plates and phage displaying the original mouse/humanchimaeric Fab or the humanised Fab fragments (this assay is notsensitive enough to allow the detection of soluble scFv fragments) (FIG.3). FIG. 3 shows results of ELISA of phage displaying Fab fragments,with one chain displayed on phage as a g3p fusion (indicated by fd- . .. ), and the other chain provided as a secreted, soluble chain partner.Clones display a combination of the following chains: fd-DOG1 (noantibody); light chains: mouse MoV_(L) (from Mab32), human VλA2 and VλC4(selected with mouse V_(H)),; heavy chains: mouse MoV_(H) (from Mab32),human V_(H) P1,2,3 (selected with human VλA2, VλD1, VλC4), human V_(H)LM2, V_(H) LM8 (selected with mouse V_(L)). Vλ chains are indicated asVL. Fully humanised clones were obtained which bound to both peptide 301and TNF.

In addition, to show that the human scFv fragments compete with theoriginal antibody for binding to TNF, we analysed binding of the scFvconstructs in a competition ELISA with the Fab fragment derived byproteolytic cleavage of Mab32. Single chain Fv fragments were incubatedon a TNF-coated surface with increasing amounts of the Fab fragment andthe amount of bound scFv detected in ELISA (FIG. 7). Each of the scFvfragments competes with the FabMab32 for binding to TNF, including boththe original scFv-Mab32 and the humanised scFv fragments.

Thus the fine specificity of Mab32 for peptide 301 of TNF is retainedthrough the humanisation process.

Affinity of binding of the selected V gene pairs:

The mouse Mab32 antibody and purified, monomeric forms of therecombinant mouse scFv-Mab32 and the human scFv antibodies VHP1-VλA2,VH}P2-VλA2 and VHP3-VλA2, were subjected to competition ELISA for thedetermination of the relative affinity for TNF. Antibodies wereincubated on a TNF-coated surface in the presence of increasing amountsof soluble TNF. All the clones showed a roughly similar decrease in theELISA signal over the same range of increasing TNF concentrations (withan IC50 in the 10 nM to 100 nM range). Although the parent Mab32 hasbeen reported to bind more strongly (affinity constant for binding tohuman TNF of 8.77×10⁹ M⁻¹) this reflects the bivalency of the wholeantibody molecule compared to the monovalency of a scFv fragment.

The Mab32 and VHP3VλA2 fragments were also analysed for bindingproperties using the Pharmacia BIAcore. TNF was indirectly immobilisedon the surface, and the binding of antibody monitored. The properties ofthe original mouse antibody were analysing two monomeric forms of Mab32.On the TNF surface, the Fab fragment from Mab32 by proteolytic cleavageand the scFv Mab32 showed very similar fast off rates (approximately10⁻² s⁻¹). The human VHP3-VλA2 antibody has an off rate in the samerange as the original scFv-Mab32. On rates for antibody proteininteractions are in the range seen for the interaction between otherproteins and their receptors, and cover a 100 fold range between 10⁴ and10⁶ M⁻¹ s⁻¹ (Mason D. W. and Williams, A. F., 1986, Kinetics of AntibodyReactions and the Analysis of Cell Surface Antigens, Blackwell, Oxford;Pecht, I., 1992 in Sela, M. (ed), Dynamic Aspects of Antibody Function,Academic Press Inc., New York, Vol. 6, pp 1-68). Assuming the on ratesof the antibody TNF interactions are typical of antibody proteininteractions, the off rate derived by the BIACore analysis is consistentwith the affinity indicated by the competition ELISA (Kd=10⁻⁷ to 10⁻⁸M).

Thus, these determinations are consistent with scFvMab32 and thehumanised scFv clone VHP3-VλA2 having a similar affinity and thus withthe retention of affinity, as well as specificity, through epitopeimprinted selection.

Selection of humanised antibodies by alternative pathway of epitopeimprinted selection; selecting human VH then VL chains:

To answer the question of whether further human chains could be selectedusing the alternative pathway, we first selected a human VH using themouse VL domain as imprinting chain. After four rounds of panning, twohuman VH domains dominated the population (VH-LM2 and VH-LM8). Both VHgenes show minimal differences compared with the germline (DP-46), whichis of the VH3 family. Compared with the VHP1, 2 and 3 sequences, VH-LM2and VH-LM8 use a germline of the same (VH3) family, and share the samecanonical loop structure for CDR1 and CDR2 (I. M. Tomlinson et al.,1992, supra; C. Chothia et al., 1992 J. Mol. Biol. 227, pp789-817).However, clones VH-LM2 and VH-LM8 use a rather long VH}-CDR3 comparedwith VH} P1, 2 and 3 (18 and 14 amino acid residues compared to 8,9 and10 residues respectively), and their respective germlines differ in 15amino-acid residues. To test the complementarity of the chains selectedon different (but complementary moulds), the VH-LM2 domain was combinedwith the human VλA2 domain either as a Fab on phage or in a scFvconfiguration. Both yielded binding combinations (FIG. 3 and results notshown). In addition, the mouse VL forms a relatively weak binder incombination with the human VHP3 (as Fab fragments on phage).

General Methods

Preparation of phage displaying Fab fragments on the surface using a tworeplicon system:

E. coli cells containing plasmid encoding the antibody chain for solubleexpression were grown at 37'C. in 10 ml 2x TY containing 100 ug/mlampicillin and 1% glucose to an OD600 of 0.5. The cells were infectedwith either 10¹¹ tranforming units (TU) prepared from the originallibrary (by growing libraries at 37'C. in 2 x TY with tetracyclin at 15ug/ml and collecting phage by two rounds of PEG precipitation), or with1 ml eluate (variable titre) from the panning experiments, by incubatingat 37'C. for 45 min without and 45 min with shaking. Subsequently thecells were inoculated into 500 ml of prewarmed medium with ampicillin(100 ug/ml) and tetracyclin (15 ug/ml), and after growing for 1 hour at37'C. transferred to a 30'C. shaker for an overnight incubation. Phagewas prepared by two rounds of PEG-NaCl precipitation, and resuspended inwater to approximately 10¹³ TU/ml before selection.

Selection of binders:

Immuno tubes (Nunc) were coated overnight with 2 ml of a 10 ug TNF perml of 50 mM bicarbonate buffer (pH 9.6). Incubation with phage, washingand elution conditions were as described by Marks et al., 1991. E. colicells expressing the soluble complementary chain were infected with theeluted phage and grown in 2 x TY medium with tetracyclin ug/ml) foramplification of the selected phage population (as described above).

Screening clones for binding:

Individual clones were assayed for binding in phage ELISA, by preparingone-chain phage stocks, and using this phage to infect cells harbouringa gene encoding a partner chain (the one(s) which was (were) used toselect the clones). Phage displaying Fab fragments prepared from such (2ml) overnight cultures as decribed above, was 10-fold concentrated byPEG precipitation and 50 ul assayed in ELISA. Recombinant human TNF-α(produced in yeast, specific activity of 3.2×10⁷ units/mg, PeptideTechnology) was coated onto plastic flat-bottomed microtiterplates(Falcon 3912) at 10 ug/ml in 50 mM bicarbonate buffer (pH 9.6) or PBS,at 50 ul/well overnight at room temperature. After washing with PBS,blocking for 2 hours with 2% Dried Skimmed Milk Powder (Marvel), 50 ulphage per well was added to 50 ul 4% Marvel, after which the ELISA wascontinued (with anti-phage antiserum) as described (Marks et al., 1991and PCT/GB91/01134). For the detection of binding to the Mab32 epitope,the 301-peptide (gift from Peptide Technology, sequenceH-VRSSSRTPSDKPVAHVVA-OH SEQ ID NO 119) was coated by overnightincubation at room temperature with 100 μl per well of 10 ug/ml peptidein PBS. The wells were blocked for 2 hours with 3% BSA, and the ELISAcontinued as above (using incubations with Marvel).

Alternatively, soluble antibody fragments were made by r ecloning theselected chains in one plasmid (either as scFv fragments), inducingexpression in the appropriate strain and detecting bound antibodyfragments in the supernatant of the E. coli cultures with theanti-myc-tag antibody. Purified monomeric scFv fragments (see below forpurification) were used for competition ELISA experiments and to checkthe specificity of binding. For the specificity ELISA, plates werecoated with various proteins as described (Marks et al., 1991, supra andWO 92/01047). In all cases where soluble fragments were assayed inELISA, Marvel was replaced by BSA.

For competition ELISA, we used Fab fragment derived by proteolyticcleavage of Mab32 (gift of Peptide Technology) and either the9E10-protein A purified mouse scFv-Mab32 or the protein A-purified humanscFv fragments (VHP1/2/3-Vλ2). To a TNF coated plate (blocked with 3%BSA in PBS), a mixture was added of one of each of the four scFvfragments and increasing amounts of the mouse Fab fragment (in 100 ultotal volume, 1.2 ul, 4 ul and 10 ul of a 1 mg/ml solution. Afterincubation for 2 hours, the plate was washed, and bound scFv detectedwith the antibody 9E10 (which recognises the C-terminal myc-tag of thescFv fragments; Munro and Pelham 1986 Cell 46 291-300) and peroxidaselabelled goat anti-mouse antibody (Fc specific). As negative control,the same antibodies were used to detect increasing amounts of Fabfragment only (the anti-mouse (Fc specific) cross-reacts weakly with themouse Fab). Experiments were done with a range of concentrations of scFvfragments (up to 250 ug/mL), to establish the minimal detectable amountof scFv where competition was visible (when an excess of scFv is used,no competition is visible).

DNA fingerprinting and sequencing of clones:

The V-gene DNA of phage clones positive in TNF-ELISA was analysed byDNA-fingerprinting (as in Clackson et al., 1991, supra). Briefly, thegenes were amplified with fd-PCR-BACK and, for heavy chains, fd-SEQ1,or, for light chains, HULAMBDASEQ, for 25 cycles (94'C., 1 min; 50'C., 1min and 72'C., 2 min), and the DNA cut with BstNI. Analysis of thedigest by agarose gelelectrophoresis allowed then to identify majordifferences between the positive clones. The nucleic acid sequences ofselected V-regions were determined by the dideoxy chain terminationmethod (F. Sanger et al., 1977 Proc. Natl. Acad. Sci. USA 74,pp5463-5467) using a Sequenase kit (USB) and appropriate sequencingprimers. Murine V-genes cloned into pHEN1 were sequenced with pHEN-SEQand LINKSEQ; human VH genes were sequenced with a primer sited in thehuman CH1(mu) region (Hu-MCH1FORSEQ2), while for human Vλ genes,HULAMBDASEQ was taken.

Recloning selected chains for soluble expression:

Selected light chains were amplified with SfiI-tagged backward and NotItagged forward primers for cloning into pUC19-pelB-myc. Human lightchains VλA2, VλD1 and VλC4 were amplified from fd with HuVL1BACKSFI andHuCL1FORAMBNOT and cloned into pUC19-pelB-myc (as SfiI-NotI fragments)for soluble expression as tag-less light chains (in non-suppressorstrains).

Alternatively, selected V-genes were assembled as scFv genes essentiallyas described (Clackson et al., 1991, supra; Marks et al., 1991, supraand WO 92/01047) using oligonucleotides described in Marks et al. (1991,supra and WO 92/01047) and cloned into pHEN1 (Hoogenboom et al., 1991,supra) for expression in HB2151 E. coli cells as soluble scFv fragmentswith a C-terminal myc-tag.

Purification of scFv fragments:

The original mouse scFv-Mab32-c14 fragment with the C-terminal myc-tagwas purified from E. coli supernatant using an affinity 9E10-Protein Acolumn (as described in Clackson et al., 1991, supra). The human scFvfragments (all with VH3 genes) were purified on Protein A-sepharosecolumns (H. R. Hoogenboom and G. Winter, J. Mol. Biol. 227, pp381-388,1992). All proteins were further purified using gelfiltration(Superdex-75, Pharmacia), and the monomeric fraction taken for BIAcoreanalysis.

Standard polymerase chain reaction

The standard reaction mixture for the polymerase chain reaction was: 5μl template DNA; 20 pmol back primer(s); 20 pmol forward primer(s); 10mM KCl; 10 mM (NH₄)₂ SO₄ ; 20 mM Tris HCl (pH88); 2 mM MgCl₂ ; 100 μgBSA/ml and 1 unit Taq DNA polymerase (Perkin Elmer Cetus). The reactionmixture was overlaid with mineral oil and subjected to 30 cycles ofamplification using a Techne thermal cycler (Duxford, England). Thecycle was 94° C. for 1 min (denaturation), 55° C. for 1 min (annealing)and 72° C. for 1.5 min (extension). Other PCR conditions are given inthe text or in references.

Purification of DNA products

Products were purified following agarose gel electrophoresis usingGeneclean (Bio101) or by electroelution and ethanol precipitation (JSambrook et al 1989 Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.)

Restriction enzyme digests

Were performed according to the manufacturers instructions (New EnglandBiolabs, CP Labs, Bishops Stortford, Herts). Ligations were performedaccording to Sambrook et al (1989 supra).

Conclusion

We have shown that a mouse antibody can be rebuilt into a human antibodywith the same specificity by the process of epitope imprinted selection(EIS).

A library of human light chains were shuffled with a mouse VH domain,binding combinations selected and then used in a second shuffle as`docking domains` for a library of human VH genes. Completely humanantibodies were isolated from such `genuine` human library. Theantibodies were shown to bind retain binding specificity. Alternatively,the mouse VL was used as docking chain for selecting human VH partners.Such VH domains can be used to find human VL genes, or, alternatively,can be combined with human VL domains selected with the mouse VH domain.Indeed, binding activity was obtained by combining two independentlyselected V-genes, pointing towards potential additivity of the EISprocedure.

The EIS approach may serve to humanise antibodies more rapidly than byCDR-grafting (Riechmann et al., 1988, supra), as this method requiresvery often a detailed knowledge of the 3-D structure of the antibody.However, the EIS method can be extended to for example antibodyrepertoires obtained by phage selection from immunised rodents.Following immunisation with antigen, a repertoire of V-genes with highaffinity and specificity may be selected and then used in a epitopeimprinted selection (see example 4) to generate a range of humanantibodies of high affinity and enriched for the desired specificity.

EXAMPLE 2 Humanising rodent antibodies using polycombinatoriallibrairies and CDR imprinting

CDR 3 of the heavy chain is generally found to be the most variable ofall CDRs in terms of both length and sequence, and can make importantcontacts with antigen (Winter, G. and Milstein C. Man-made Antibodies.(1991) Nature 349, pp293-299). This is an important consideration whenhumanising, whether by CDR-grafting (Patent GB2188638B) or as describedin this application using chain-shuffling. It may be advantageous toapply the polycombinatorial approach to humanising by a chain-shufflingprocess in which the VHCDR3 sequence of the rodent antibody is imprintedupon the human VH segments.

In the polycombinatorial approach, one fragment eg VHCH1, is displayedon phage as a fusion from a phage vector and the other, eg light chain,is expressed as a soluble fragment from eg a plasmid. Super-infectionwith phage is used to display both fragments on phage as a Fab fragment.

In this example a mouse anti-HIVgp120 monoclonal antibody was humanised.The VH domain of this mouse antibody was fused to the human Cgammaldomain and cloned into pUC19Sfi/Not/polymyc.

A repertoire of naive human light chains cloned as g3 fusions infd-DOG-1 (also known as fd-CAT-2 in WO 92/01047) was then infected intothe cells carrying the chimaeric heavy chain, and phage selected onantigen. These phage have both heavy and light chains on their surface,though the phage genome encodes just the light chain; this is not aproblem since the only heavy chain is the one provided.

Light chains selected this way were then paired with a library of naivehuman VH domains, PCR-amplified in such a way that CDR3 of the humanantibodies were replaced with that of the original mouse heavy chain.

Section (a) deals with construction of a chimaeric Fab fragment in whichthe mouse F58 VH and VL domains are fused to human CH1 and CK sequences.This clone was used in early characterisation of the antibody and servedas a template for subsequent PCR amplification of the heavy chain, whichwas then cloned into pUC19 Sfi-Not polymyc as a PstI-Not I fragment(section (b)). Section (c) describes construction of a human light chainrepertoire in fdDOG, which was then infected into cells containing thechimaeric heavy chain on pUC19 (section (d)). The resulting phage werepanned against the peptide (section (e)) and the selected light chainsPCR-amplified and cloned as Asc I-Not I fragments alongside thechimaeric heavy chain (section (f)) and assayed for their ability tobind antigen by ELISA (section (g)). Selected light chains were reclonedin pUC (section (h)) and naive human VH domains amplified with amutagenic primer imposing the F58 CDR3 sequence on the domains, and theresulting fragments cloned in phage (section (i)). This repertoire ofimprinted heavy chain phage was then used to infect cells carrying theselected light chains on pUC and the resulting phage panned on antigen.Finally, the selected heavy and light chains are cloned together on thesame replicon and assayed for binding to antigen (section (j)).

(a) Cloning of F58 chimaeric heavy chain

(i) cDNA synthesis and primary PCR.

Five ml of cultured hybridoma cells (approximately 2×10⁶ cells) werewashed in PBS, pelleted, resuspended in 200 ul 0.1% diethylpyrocarbonatein water and immediately boiled for 5 minutes. After centrifugation, 68ul of the `boilate` supernatant was added to a 28 ul reaction mixtureresulting in a 96 ul reaction mixture containing 140 mM KCl, 50 mMTris.HCl (pH8.1@42'C.), 8 mM MgCl₂, 10 mM DTT, 500 uM deoxythymidinetriphosphate, 500 uM deoxycytidine triphosphate, 500 uM deoxyadeninetriphosphate and 500 uM deoxyguanine triphosphate nucleotidetriphosphate (500 uM dNTPs), 160 units of human placental RNAseinhibitor and 10 pmol of forward primer. Four ul (100 units) of avianmyeloblastosis virus (AMV) reverse transcriptase was added, the reactionincubated at 42'C. for 1 hour, heated to 100'C. for 3 minutes, quenchedon ice and centrifuged for 5 minutes. The supernatant was then usedimmediately for PCR.

Separate PCR amplifications were performed for the heavy and lightchains. Fifty ul reaction mixtures were prepared containing 5 ul of thesupernatant from the cDNA synthesis, 250 uM dNTPs, 50 mM KCl, 100 mMTris.HCl (pH 8.3), 1.5 mM MgCl₂, 175 ug/ml BSA, 20 pmol each of theappropriate mixtures of forward and back primers (Clackson, T et al.(1991) supra.) and 1 ul (5 units) Thermus aquaticus (Taq) DNA polymerase(Cetus, Emeryville, Calif.). The reaction mixture was overlaid withparaffin oil and subjected to 30 cycles of amplification using a TechnePHC-2 thermal cycler. The cycle was 94'C. for 1 minute (denaturation),55'C. for 1 minute (annealing) and 72'C. for 1 minute (extension). Theproduct was analyzed by running 5 ul on a 2% agarose gel. The remainderwas extracted twice with ether, once with phenol/chloroform, ethanolprecipitated and resuspended in 50 ul of water.

(ii) Cloning and sequencing of amplified VH and Vk DNA.

The amplified VH DNA was digested with PstI and BstEII, purified on a 2%low melting point agarose gel and ligated into M13VHPCR1 digested withPstI and BstEII (Orlandi, R., D. H. Gussow, P. T. Jones and G. Winter.(1989). Cloning immunoglobulin variable domains for expression by thepolymerase chain reaction. Proc. Natl. Acad. Sci., USA, 86 (10),pp3833-7). The amplified VK DNA was digested with PvuII and Bgl II andligated into M13VKPCR1 digested with Pvu II and Bcl I. The ligationmixtures were used to transform competent TG1 cells. Two clones fromseparate amplifications were sequenced for each VH and Vk chain usingthe dideoxynucleotide chain termination method.

(iii) Generation of an Fab construct for expression in E. coli

A chimaeric Fab containing the F58 variable domains and the human IgG1CH1 and Ck domains was constructed by ligating the F58 V-domains into avector containing the constant domains. M13VHPCR1 containing the F58 VHwas digested with PstI and BstEII. The resulting F58VH fragment was thenpurified on a 1.5% agarose gel, isolated from the gel with Geneclean andligated into pJM-1FabD1.3 (PCT/GB91/01134) digested with PstI andBstEII. The ligation mixture was used to transform competent E. coliN4830-1 cells (Gottesman, M. E., Adhya, S. and Das, A. (1980) J. Mol.Biol. 140, pp57-75) and clones containing the F58 VH identified byrestriction analysis of RF DNA. The F58 Vk was amplified by PCR with theprimers Vk2BACK and Vk3FOR2 (Clackson, T. et al. (1991) supra.) usingM13VkPCR containing the F58 Vk as template. The PCR product was digestedwith SacI and XhoI, purified on a 1.5% agarose gel, isolated from thegel with Geneclean and ligated into pJM-1 Fab vector containing the F58VH digested with SacI and XhoI. The ligation mixture was used totransform competent E. coli N4830-1 cells and clones containing the F58Vk identified by restriction analysis of RF DNA.

(b) PCR and cloning of F58 chimaeric heavy chain

The F58 chimaeric heavy chain derived above was PCR amplified from F58Fab clone DNA, using the primers VH1BACKSFI15 and HUCH1FORASCNOT (Table1). The resulting ca. 700 bp fragment was digested with Pst I and Not Iand cloned into Pst I and Not I-cut pUC19Sfi/Not/polymyc plasmid usingstandard procedures (Sambrook, J. et al. 1989, supra. and example 1).

(c) Construction of light chain repertoire

Kappa and lambda-chain genes were amplified separately using anequimolar mixture of the appropriate family based BACK and FORWARDprimers (Table 1). Kappa-chain genes were amplified from the cDNAsynthesis using HUCKFOR primer, using an equimolar mixture of the 6HUVKBACK 1a-6a primers in conjunction with the HUCKFORSER primer.Lambda-chain genes were amplified from the cDNA synthesis using theHUCLFOR primer, and amplified using an equimolar mixture of the 7HULBACK 1-6 primers -in conjunction with the HUCLFORSER primer. In eachcase 50 μl reaction mixtures were prepared containing 5 μl of thesupernatant from the appropriate cDNA synthesis, 20 pmol totalconcentration of the BACK primers, 20 pmol total concentration of theFORWARD primers, 250 μM dNTPs, 10 mM KCl, 10 mM (NH₄)₂ SO₄, 20 mMTris.HCl (pH 8.8), 2.0 mM MgCl₂, 100 mg/ml BSA and 1 μl (1 unit) VentDNA polymerase (New England Biolabs). The reaction mixture was overlaidwith mineral (paraffin) oil and subjected to 30 cycles of amplificationusing a Techne thermal cycler. The cycle was 94° C. for 1 minute(denaturation), 57° C. for 1 minute (annealing) and 72° C. for 2.5minutes (extension). The products were purified on a 2% agarose gel,isolated from the gel by Geneclean (Bio-101) and resuspended in 25 μl ofH₂ O.

Two different pullthrough reactions were now performed on each of thetwo light chain preparations. Kappa-chain genes were amplified in tworeactions, using an equimolar mixture of the 6 HUVKBAAPA primers inconjunction with HUCKFORSERNOT. Lambda-chain genes were also amplifiedin two reactions, using an equimolar mixture of the 7 HUVLBAAPA primersin conjunction with HUCLFORSERNOT. Pullthrough conditions were performedas for the primary light chain PCRs above except that 25 cycles ofamplification were used.

PCR products were digested with Apa LI and Not I using the standardformat and cloned into Apa LI and Not I-cut fd-DOG as described inexample 1. Phage were prepared from the library clones as described inexample 1 and used to infect cells containing the heavy chain (seebelow).

(d) Production of shuffled Fab phase.

Cells containing the F58 chimaeric heavy chain were grown overnight at37'C. in 2YTAG and 500 ul added to 50 mls fresh 2YTAmp medium, prewarmedto 37'C., in a conical flask. The cells were grown with shaking to OD600of about 0.5 before adding a total of 10¹¹ phage from the light chainrepertoire. The culture was left at 37'C. for 45 minutes without shakingthen shaken vigorously for another 45 minutes at 37'C. before addingtetracyline to 15 ug/ml and shaking overnight.

Phage were harvested by PEG precipitation of the culture supernatant aspreviously described (example 1) and used for selection experiments.

(e) Panning of shuffled Fab phage.

This was performed on maxisorb plates as previously described (Marks 3.et al., 1991, supra.), with the exception that the Tubes were coatedwith env gp120 V3 loop peptide of HIV-1 isolate IIIB dissolved to 10ug/ml in water. This peptide was obtained from the AIDS-directed program(repository ref: ADP737) and has the sequence:CTRPNNNTRRSIRIQRGPGRAFVTIGKIGNMRQAHCN (SEQ ID NO 120) The phage elutedfrom the tubes were used to re-infect fresh cells containing the F58chimaeric heavy chain and the panning/reinfection procedure repeatedanother three times.

(f) Recloning of selected light chains.

Selected light chains were PCR-amplified from fd-DOG Light chain DNAusing primers G3LASCGTGBACK and HUCLFORSERNOT or HUCKFORSERNOT. TheG3LASCGTGBACK primer anneals upstream of the translational start of thegIII signal in fd, and brings in an Asc I site and a ribosome bindingsite (RBS). These fragments were digested with Asc I and Not I andcloned into Asc I and Not I-cleaved pUC19F58 plasmid (as shown in FIG.8) so as to create a cistron, enabling soluble Fab to be produced. Thiswas analysed for peptide binding in ELISA and bound antibody detected byvirtue of the myc tag peptide on the end of the light chain.

(g) ELISA

This was performed as described below.

1 Inoculate 100 ul 2 x TY, 100 μg/ml ampicillin, 1% glucose in 96-wellplates (`cell \tab wells`, Nuclon) and grow with shaking (300 r.p.m.)overnight at 37'C.

2 Use a 96-well transfer device to transfer small inocula from thisplate to a second 96-well plate containing 200 ul fresh 2 x TY, 100ug/ml ampicillin, 0.1% glucose per well. Grow at 37'C., shaking untilOD600 nm is approximately 0.9 (about 3 hrs). To the wells of theoriginal plate, add 25 ul 60% glycerol per well and store at -70'C.

3 Add 25 ul 2 x TY, 100 ug/ml ampicillin, 9 mM IPTG (final concentration1 mM IPTG). Continue shaking at 30'C. for a further 16 to 24 hrs.

4 Spin 4,000 r.p.m. for 10 min and use 100 ul supernatant in ELISA.

5 Coat plate (Falcon 3912) with 50 ul per well of peptide at 10 ug/ml inwater. Leave overnight at room temp.

6 Rinse wells 3× with PBS, and block with 200 ul per well of 1%BSA/PBS,for 1 hr at 37'C.

7 Rinse wells 3× with PBS, then add 25 ul 6% BSA/PBS to all wells.

8 Add 100 ul culture supernatant containing soluble Fab to theappropriate wells. Mix, leave 1.5 hrs room temp.

9 Discard test solution, and wash out wells 3 times with PBS, 0.05%Tween 20 and 3 times with PBS. Pipette 100 ul of 4 ug/ml purified 9E10antibody, in 2% Marvel/PBS, into each well. Incubate at room temp. for1.5 hrs.

10 Discard 9E10 antibody, and wash out wells with 3 times with PBS,0.05% Tween 20 and 3 times with PBS. Pipette 100 ul of 1:500 dilution ofanti-mouse antibody (peroxidase-conjugated anti-mouse immunoglobulins,Dakopats/ICN, or peroxidase-conjugated anti-mouse IgG, Fc-specific,Sigma. A-2554). Incubate at room temp. for 1.5 hrs.

11 Discard 2nd antibody, and wash wells 3 times with PBS, 0.05% Tween 20and 3 times with PBS.

12 Add one 10 mg ABTS (2,2'-azino bis(3-ethylbenzthiazoline-6-sulphonicacid), diammonium salt) tablet to 20 ml 50 mM citrate buffer, pH4.5. (50mM citrate buffer, pH4.5 is made by mixing equal volumes 50 mM trisodiumcitrate and 50 mM citric acid).

13 Add 2 ul 30% hydrogen peroxide to the above solution immediatelybefore dispensing.

14 Add 100 ul of the above solution to each well. Leave room temp. 20-30min.

15 Quench by adding 50 ul 3.2 mg/ml sodium fluoride. Read at 405 nm.

Note 1: Alternatively, inoculate clones from transformation plate into100 ul 2 x TY, 100 ug/ml ampicillin, 0.1% glucose in 96-well plates(`cell wells`, Nuclon) and grow with shaking (300 r.p.m.) 37'C., shakinguntil OD600 nm is approximately 0.9 (about 6 hrs). Continue with step 3.

Note 2: This method is based on that of DeBellis D. and Schwartz I.,1990 Nucl. Acids Res. 18: p1311, and relies on the low levels of glucosepresent in the starting medium being metabolised by the time the inducer(IPTG) is added.

Note 3: `Marvel` is dried milk powder. PBS is 5.84 g NaCl, 4.72 g Na₂HPO₄ and 2.64 g NaH₂ PO₄.2H₂ O, pH 7.2, in 1 liter. BSA is Bovine SerumAlbumin.

(h) Subcloning selected light chains.

Light chains were PCR-amplified from DNA of selected clones using anequimolar mix of the 7 HUVLBAS FI primers in conjunction withHUCLFORSERASCNOT or an equimolar mix of the 6 HUVKBASFIprimers inconjunction with HUCKFORSERASCNOT. Reaction conditions were as describedfor the primary PCR reactions above except that 25 cycles ofamplification were used. PCR fragments were cut with Sfi I and Not I andcloned into Sfi I and Not I-cut pUC19Sfi/Not/polym yc then transformedinto TG1.

The panning/infection process described above is essentially repeatedagain, this time with the positions of the heavy and light chainsreversed.

(i) CDR-imprinting VH

VH domains were amplified from the pooled primary PCR material preparedas described below.

Two preparations of PCR-amplified VH genes were made. Both preparationsused an equimolar mixture of the HUJHFOR primers (Table 1); in one ofthe preparations, 6 separate PCR amplifications were performed with eachof the HUVHBACK primers individually (Table 1), and in the other, asingle PCR reaction was performed with an equimolar mix of all 6HUVHBACK primers. For all seven PCRs, 50 μl reaction mixtures wereprepared containing 5 μl of the supernatant from the cDNA synthesisusing the HUIGMFOR primer, 20 pmol total concentration of the BACKprimers, 20 pmol total concentration of the FORWARD primers, 250 μMdNTPs, 10 mM KCl, 10 mM (NH₄)₂ SO₄, 20 mM Tris.HCl (pH 8.8), 2.0 mMMgCl2, 100 mg/ml BSA and 1 μl (1 units) Vent DNA polymerase (New EnglandBiolabs). The reaction mixture was overlaid with mineral (paraffin) oiland subjected to 30 cycles of amplification using a Techne PHC-2 thermalcycler. The cycle was 94° C. for 1 minute (denaturation), 57° C. for 1minute (annealing) and 72° C. for 1 minute (extension). The productswere purified on a 1.5% agarose gel, isolated from the gel by Geneclean(Bio-101) and resuspended in 25 μl of H₂ O. The seven products were thenpooled and `pullthrough` PCR reactions performed.

Six separate pullthrough reactions were performed with the mutagenicF58GRAFTJH4SAL primer and each of the HUVHBAAPA1-6 primers individually.

Pullthrough reactions were set up with the primers HUVHBAAPA (equimolarmix of all 6 primers) and HUJHFORSAL (equimolar mix of all 4 primers).50 μl reactions of containing 5 μl of the pooled PCR products from theprevious step were amplified using the same conditions as for theprimary PCR except that 25 cycles of amplification were used, and theannealing temperature was 45° C.

The resulting VH fragments were pooled and cut with Apa LI and Sal Iusing standard conditions and cloned into Apa LI and Xho I-cutfd-DOG-G1CH1 using standard protocols (Sal1 and Xho I produce compatibleCTAG overhangs). Phage were prepared from this library as describedabove, this time using the heavy chain phage to infect cells carryingthe selected light chains expressed in pUC19Sfi/Not/polymyc.

(j) Screening of final heavy-light combinations.

The end result of this process is a pool of selected heavy chains and apool of selected light chains. These are now combined at random. Heavychain clones are now PCR-amplified using an equimolar mix of all 6HUVHBACKSFI primers in conjunction HUCH1FORA SCNOT using the proceduredescribed in example 1. These fragments are cut with Sfi I and Asc I andgel-purified using standard procedures (Example 1), then ligated toequimolar quantities of Asc I-Not I -cut light chains produced in stepf) above and Sfi I and Not I-cut pUC 19Sfi/Not/polymyc vector, alsoproduced earlier. Alternatively, these Sfi I-Asc I fragments replace theF58 heavy chain in the constructs shown at the end of FIG. 8 (A). Theseconstructs were then transformed into TG1 and analysed for peptidebinding activity by ELISA as described above.

The end-products are completely human Fab fragments With the same orsimilar antigen-specificity as the parent rodent antibody.

EXAMPLE 3

Humanising rodent antibodies using random combinatorial phage displaylibraries and CDR grafting (CDR imprinted selection)

In the work described in this example the mouse anti-HIV-1 gp120monoclonal antibody F58 was humanised as shown schematically in FIG. 9.A vector (pHENMBF58VL) was constructed carrying the F58 Vk-domain, thesingle chain Fv linker and the first residues of a human VH-framework 4with a Xhol restriction site. This vector allows the cloning of VHrepertoires as Nco I/Sal I fragments to give complete scFvs. A humanrepertoire of VHs was amplified by PCR in such a way that each heavychain contained the CDR3 of the original mouse mab F58 and a humanframework 4 and was finally cloned into pHENMBF58VL. The scFv librarywas selected for binding to a HIV-1 gp120 derived peptide. Heavy chainvariable domains from selected scFvs were then combined by PCR-assemblywith a repertoire of human light chain variable domains to give fullyhumanised scFvs and again selected for antigen binding. The procedureillustrated here is therefore a synthesis of CDR-grafting andcombinatorial phage display libraries.

1. PCR amplification, cloning and sequencing of the F58 variable domains

were described in example 2. Standard PCR conditions are as in example1.

2. Construction of the F58 scFv

The VH and VL domains characterised above were assembled to form, scFvfragments, basically as described by Clackson et al, 1991, supra.

Sequences of the primers mentioned in section 2.1 are given in Clacksonet al (1991 supra) except for VH1BACKSfi which is described inHoogenboom et al, supra.

2.1 PCR amplification of the F58 VH and VL and the PCR-assemblyprocedure

About 1 ng of M13VHPCR1 (Orlandi et al, 1989 supra) containing the F58VH and 1 ng of M13VkPCR (Orlandi et al, 1989 supra) containing the F58Vk were used as template in two separate, but in terms of cyclingConditions identical, PCR amplifications: Two 50 ul reactions wereprepared containing 1 ng of either the above mentioned templates, 20pmol VH1BACK and 20 pmol VH1FOR-1 (20 pmol VK2BACK and 20 pmol VK4FOR toamplify the VL), 25.0 uM dNTPs, 5 ul 10xVent polymerase reaction buffer(as obtained from the supplier New England Biolabs) and 2 units Vent DNApolymerase. The amplifications were performed with 25 cycles of PCR(94'C. for 1 min, 55'C. for 1 min, 72'C. for 2 min).

The linker DNA was similarly amplified from about 1 ng of template DNApSW2scD1.3 (McCafferty et al., Nature 348, 552-554, 1990) using theprimers LINKBACK and LINKFOR (Clackson et al., 1991, supra).

After gel purification 1 ug each of the VH and the Vk product were mixedwith 300 ng of linker in a 25 ul PCR mix without primers. and cycled 7times (94'C. 2 min, 72'C. 4 min) to join the fragments, then amplifiedfor 20 cycles (94'C. 1.5 min, 72'C. 2.5 min) using 25 pmol each ofVH1BACK and VK4FOR primers. Finally, the assembled product was gelpurified and reamplified with the primers VH1BACK-SfiI and VK4FOR-NotIto append restriction sites (Note VK4FOR and VK4FOR-NotI are equimolarmixture of 4 different primers). The F58 scFv was then digested withSfiI and NotI and cloned into the similarly digested vectorpUC119Sfi-Notpolymyc. Recombinants were subsequently verified by DNAsequencing.

3. Construction of the vector pHENMBF58VL

10 ng of purified pUC119F58scFv DNA from the clone isolated above wasused as template in a standard PCR amplification with the primersHuVHLBACK-XhoI and VK4FOR-NotI. The PCR product was purified, digestedwith Xhol and Not1 and cloned into the similarly digested vector pHEN1to generate the new vector pHENMBF58VL.

4. Amplification and cloning of the human heavy chain repertoire

4.1. Preparation of the cDNA template

The procedure was exactly as described in example 2(a)(i) using the IgMconstant region forward primers described by Marks et al, 1991, supraand in WO 92/01047.

4.2. PCR of Heavy chain variable domains

The 6 separate reactions to amplify the heavy chain variable domainscontained each 2 ul of the cDNA synthesis from above, 20 pmol of theindividual HUVHBACKSfi primers and 20 pmol of the primer F58GRAFTSAL inan otherwise standard PCR mixture. Since the primer F58GRAF TSAL had toanneal to the framework 3 regions of the 6 human VH families, theannealing temperature for the PCR cycling was reduced to 45'C. The PCRwas run for 25 cycles (94'C. 1 min, 45'C. 1 min, 72'C. 1 min), afterwhich the products were gel purified. To obtain more material for thesubsequent cloning steps a secondary PCR was performed using the sameseries of HuVHBACKSfi primers and the primer HuJH4FORSalI, theconditions were as above with the exception of an annealing step at52'C. instead of 45'C.

5. Generation of the scFv library

Equal amounts each of the PCR products from above were pooled anddigested with NCoI and SalI. 1 ug of this material was ligated into3 ugof NcoI/SalI digested vector pHENMBF58VL. The Ligation reaction wasprocessed and used for the transformation of TG1 cells as described inHoogenboom et al supra and WO 92/01047.

6. Selection of the scFv library for antigen binders

The infection of the library in TG1 with helper phage VCSM13 and theprocessing of the phages for the subsequent rounds of selection was doneas described (Hoogenboom et al, 1991, supra and WO 92/01047). For eachselection, 8 wells of a Maxisorp plate (NUNC) were inoculated with a 50ul solution of 3 ug/ml peptide in water. The wells were allowed to drycompletely over night, blocked with 1% BSA in PBS for 30 min at roomtemperature and finally washed 2× with PBS. To each well were added 50ul of phage and 150 ul of Tris-HCl, pH 8.3, 0.5M NaCl, 4% BSA. After 2hrs the wells were washed 3× with Tris-HCl, pH 8.3, 0.1M NaCl and 3×with Tris-HCl, pH 8.3, 0.5M NaCl. Elution of bound phages and infectionTG1 cells was performed as described (WO 92/01047). Briefly: Phages wereeluted with 50 ul of 100 mM Triethylamine per well for 10 min. Theeluted material was than immediately neutralized with 1/2 volume of 1MTris-HCl, pH 7.4 and used to infect logarithmically growing TG1 cell.

The peptide antigen corresponds to the gp120 V3 loop of the HIV-1isolate IIIB and was obtained from the AIDS-directed program of the MRC(repository reference ADP737). The sequence is:

    YCTRPNNNTRRSIRIQRGPGRAFVTIGKIGNMRQAHCY (SEQ ID NO 121)

7. Analysis of selected scFvs

After 3 rounds of selection an aliquot of the eluted phages was used toinfect HB2151 cells to allow for expression of soluble scFvs. Theidentification of individual binding clones by peptide. ELISA was doneas described previously in example 2 except that scFv fragments ratherthan Fab fragments were prepared.

About 15% of the analysed clones scored positive at this stage, givingELISA signals of 50%-150% of the signal obtained with the original F58scFv which was run as a control on the same plate (see FIG. 10). None ofthe positive scFvs reacted with plastic, Hen eggwhite lysozyme or humanthyroglobulin.

Nucleotide sequence analysis revealed that the positive clones containedsomatically mutated human VH-genes derived from germ line genes of theVH-families 1 and 6 (see FIG. 11).

8. Replacing the F58 light chain variable domain by a repertoire ofhuman light chain variable domains

About 10 ng of the pooled clones, which have been shown to be active inthe peptide ELISA, were used as template in a 50 ul standard PCRamplification to obtain the selected heavy chain variable domains. Asprimers were used 20 pmol of each LMB3 and HuJH4FORASSXhoI. The humanlight chain variable domains were amplified from an IgG-scFv library (WO92/01047 example 42) using 50 ng of DNA as template with the primersfdSEQ1 and HuVHLBACK in a standard PCR amplification. The products ofboth heavy and light chain amplifications were gel purified and used ina PCR assembly (1 ug each) for 7 cycles essentially as described above.Cycling conditions were 94'C. 1 min, 55'C. 1 min and 72'C. 2 min. Aftercompleting the assembly reaction the primers LMB3 and fdSEQ1 were added(20 pmol each) and the PCR was continued for further 20 cycles. Theassembled products were gel purified and digested with NcoI and NotI.Finally 1 ug of cut scFvs were ligated into 3 ug of the NcoI/NotIdigested vector pHEN1. The resulting library scFv was selected asdescribed above (step 6). Finally individual clones were screened forbinding to the V3 loop peptide as mentioned before (step 7).

The end products are completely human scFv fragments with the same orvery similar specificity as the parent murine antibody F58, ie they bindto gp120 V3 loop but not to heterologous proteins.

EXAMPLE 4

Isolation of human antibodies derived from the immune repertoire of animmunised mouse, by epitope imprinted selection

This example describes the generation of human antibodies specific forTNF-alpha from an original library of mouse antibodies derived from animmunised mouse by successive rounds of chain shuffling, illustrating afurther embodiment of the present invention. The method depends upon thefact that an intermediate VH/VL combination, where one chain is mouseand the other is human, binds to the antigenic epitope to which theoriginal mouse VH/VL pairings are directed.

In this example, it is preferable to increase the frequency ofantibodies to the antigen, here TNF-alpha, in the mouse library bypreselecting the mouse library for binding to the antigen. Thispopulation of mouse antibodies can then be humanised by chain shuffling.The VH chains from the selected mouse population are kept fixed andassembled with a set of human VL chains from a naive human library. Thismouse VH/human VL library is then subjected to rounds of selection forbinders to the antigen of interest. This selected population ofsemi-humanised antibodies is then fully humanised by pairing these humanVLs with human VHs again derived from a naive human library. Theresulting library can then be selected for binders specific for theantigen. Conversely, in the first instance mouse VL chains can be fixedand assembled with human VH chains, and after selection for binding toantigen fully humanised by pairing the isolated human VH chains with VLfrom the naive human library.

1. Preparation of single chain Fv phage display library from animmunised mouse

RNA is extracted from lymphocytes prepared from the spleen of a mousehyperimmunised with TNF-alpha. DNA sequences encoding variable regionsof the heavy and light chains are amplified by PCR and assembled assingle chain Fv fragments as described in WO 92/01047. Sites areincorporated into the 5' and 3' ends for restriction endonucleases ApaL1and Not1. Following digestion with ApaL1 and Not1 the assembled DNA isligated into ApaL1/Not1 digested pCANTAB3-myc (FIG. 12) andelectroporated into E. coli TG1 cells and plated on ampicillin selectionmedia (see protocol (h) below).

2. Selection of phage displaying mouse scFv fragments binding to TNF.

Phagemids are rescued by superinfection with M13KO7 helper phage andprecipitated using 1/5 volume 20% polyethylene glycol/2.5M NaCl. Aselected population of TNF-alpha binding phage antibodies is prepared bypanning the phage antibodies on TNF-alpha coated immunosorb tubes (1 mL,10 ug/mL in PBS) and eluting with 1 mL 100 mM triethylamine for 5minutes followed by neutralisation with 0.5 mL TRIS (1M, pH 7.4).

The eluted phage is used to infect 10 mL TG1 cells in logarithmic growthfor 30 minutes at 37'C. and plated for ampicillin resistant colonies.The resulting colonies are scraped into 2YT media and rescued withM13KO7. Phage antibodies are selected once again on TNF-alpha coatedimmunosorb tubes as above. E. coli TG1 cells are infected with theeluted phage and plated and ampicillin resistant colonies are scrapedinto 2YT media and glycerol added to 15% and stored at -70'C. Thefrequency of clones binding to TNF in the selected population is checkedat this stage by ELISA as described in example 1.

3. Preparation of DNA as source of fragments for chain shuffling

Human heavy and light chain DNA

A library encoding scFv fragments derived from the V genes ofunimmunised humans has been constructed in the vector pHEN1 (WO92/01047, example 42).

DNA preparation

Miniprep DNA was prepared by alkaline lysis from the colonies of thesecond round of selection (mouse DNA) and from the naive human library(human DNA).

Approximately 5×10⁷ cells from glycerol stocks are grown overnight at30'C. in 10 mL 2YT media containing 100 ug/mL ampicillin, 2% glucose.The cells are pelleted by centrifugation (3500 rpm, 15 minutes) and DNAprepared from the cell pellet by alkaline lysis using a commerciallyavailable kit (Promega).

4. Preparation of DNA encoding chain shuffled scFv fragments

The following primers are used:

    ______________________________________                                        LMB3     5'CAG GAA ACA GCT ATG AC 3'                                                                          SEQ ID                                                                        NO: 122                                       FDTSEQ1  5'GTC GTC TTT CCA GAC GTT                                                                            SEQ ID                                                 AGT 3'                 NO: 123                                       PCRHLINK 5'ACC GCC AGA GCC ACC TCC                                                                            SEQ ID                                                 GCC 3'                 NO: 124                                       LINKPCRL 5'GGC GGA GGT GGC TCT GGC                                                                            SEQ ID                                                 GGT 3                  NO: 125                                       ______________________________________                                    

LMB3 and FDTSEQ1 anneal, respectively, to sequences 5' and 3' flankingthe antibody cloning sites of the pCANTAB series of vectors. PCRHLINKand LINKPCRL and anneal with the sequence encoding the (gly4ser3)peptide linker. Using the primers in pairs LMB3/PCRHLINK andFDTSEQ1/LINKPCRL in PCT amplifications, heavy and light chain fragmentswill be generated that have complementary 3'/5' ends and restrictionrecognition sites at the 5'/3' ends.

In this procedure the human library described by Marks et al., 1991(supra) is used as the source of heavy chain and light chain variabledomain encoding DNA. The following chain shuffling protocol is used foreach shuffle. It is followed once for, e.g. shuffling human naive lightchain variable domains with the mouse heavy chain variable domains andthen again with the selected human light chain variable domains with thehuman naive heavy chain variable domains. The DNA encoding shuffled scFvfragments produced by this procedure is inserted into pCANTAB3-myc orpCANTAB5-myc (FIG. 13) depending on whether mouse VH genes or human VHgenes have been used in the shuffle respectively.

(a) Primary PCR

Prepare primary heavy and light chain products in the followingreactions:

    ______________________________________                                        HEAVY            LIGHT                                                        ______________________________________                                        Miniprep DNA (e.g.                                                                         2     ng    Miniprep DNA (e.g.                                                                         2   ng                                  mouse)                   human)                                               LMB3 primer (10                                                                            2.5   μl FDTSEQ1 primer (10                                                                         2.5 μl                               pmoles/μl)            pmoles/μl)                                        PCRHLINK primer                                                                            2.5   μl LINKPCRL primer                                                                            2.5 μl                               (10 pmoles/μl)        (10 pmoles/μl)                                    10X PCR reaction                                                                           5.0   μl 10X PCR reaction                                                                           5.0 μl                               buffer.sup.1             buffer.sup.1                                         5 mM each dNTP's                                                                           2.5   μl 5 mM each dNTP's                                                                           2.5 μl                               Taq polmerase (5 U/                                                                        0.3   μl Taq polymerase (5 U/                                                                       0.3 μl                               μl)                   μl)                                               water        37    μl water        37  μl                               ______________________________________                                    

The "reaction buffer" is 10X PCR reaction buffer: 0.1M TRIS pH 8.3, 0.5MKCl, 15 mM MgCl₂, 0.1% gelatine. PCR conditions are 25 cycles of 94'C. 1minute, 60'C. 1 minute, 72'C. 2 minutes with a final 10 minutes at 72'C.

(b) Product purification

Primary PCR products are purified on agarose gels and purified using 5ul of "glass milk" (Geneclean, Bio 101) with two elutions in water of 10ul each.

(c) Assembly by PCR

Assembly is carried out as follows:

    ______________________________________                                        purified heavy    2.5        ul                                               purified light    2.5        ul                                               10X reaction buffer                                                                             2          ul                                               5 mM each dNTP's  1.0        ul                                               Taq polymerase    0.2        ul                                               water             37         ul                                               ______________________________________                                    

PCR conditions are 25 cycles of 94'C. 1 minute and 65'C. for 4 minutes,followed by a final 10 minutes at 72'C. The PCR conditions given for theprimary PCR will also work, but 65'C. is preferred for the linkagereaction.

(d) Secondary PCR

For secondary PCRs use 1 ul of the linked material from (c) as template.Set the reaction up as follows:

    ______________________________________                                        linked PCR product    1         μl                                         LMB3 primer 10 pmoles/μl)                                                                        2.5       μl                                         FDTSEQ1 primer (10 pmoles/μl)                                                                    2.5       μl                                         10X PCR reaction buffer                                                                             5.0       μl                                         5 mM each dNTP's      2.5       μl                                         Taq polmerase (5 U/μl)                                                                           0.3       μl                                         water                 37        μl                                         ______________________________________                                    

PCR conditions are 25 cycles of 94'C. 1 minute, 60'C. 1 minute, 72'C. 2minutes with a final 10 minutes at 72'C.

(e) Digestion of secondary PCR product (insert)

Extract the secondary PCR product with phenol:chloroform and ethanolprecipitate, to remove Taq polymerase. Dissolve the PCR product in 270ul of water, add 30 ul of 10X reaction buffer. Add 50 units Not1 anddigest at 37'C. for 3 hours. Extract with phenol:chloroform and ethanolprecipitate. Resuspend the pellet in 270 uL water, and add 30 uL 10Xreaction buffer. If the VH used is human use Sfi1 (50 units) and digestovernight at 50'C., whereas if the VH is mouse use ApaL1 (50 units) anddigest overnight at 37'C.

10X ApaL1 buffer: 0.5M potassium acetate, 0.2M TRIS-acetate, 100 mMmagnesium acetate, 10 mM dithiothreitol (pH 7.9@25'C.).

10X Not1 buffer: 1.5M NaCl, 100 mM TRIS-HCl, 100 mM MgCl₂, 0.1% TritonX-100 (pH 7.9@25'C.).

10X Sfi1 buffer: 0.5M NaCl, 100 mM TRIS-HCl, 100 mM MgCl₂, 10 mMdithiothreitol (pH 7.9@25'C.).

(f) Purification of digested product

The digest is treated with phenol:chloroform, ethanol precipitated anddissolved in 20 ul water. The product is run out on a 1.5% agarose gel,the band at approximately 750 bp is excised from the gel and purifiedusing a geneclean kit. The DNA is eluted into a final volume of 10-15 ulwater and is ready for cloning.

5. Preparation of vector DNA and cloning Preparation of vector DNA

Plasmid DNA is prepared by the alkaline lysis method and is purified bycesium chloride centrifugation. The purified DNA is digested using a DNAconcentration of 100 ug/ml with Sfi1 (for pCANTAB5-myc) or ApaL1 (forpCANTAB3-myc) according to manufacturers instructions (50'C. for Sfi1,overnight if convenient) followed by a 3 hour digestion with NOt 1. Thedigestion product is loaded on directly on to a Chromaspin 1000 columnto remove the stuffer fragment and spun for 3 minutes at 2200 r.p.m. ina bench top centrifuge. The DNA was then phenol:chloroform extracted anddissolved at 100 ug/ml for use.

Ligation and transformation

Ligations are carried out using an Amersham ligation kit as follows:

    ______________________________________                                        Vector DNA           1      μl (100 ng)                                    insert DNA           2      μl (10-50 ng)                                  10 mMMgCl, 200 mM Tris pH 7.4                                                                      3      μl                                             buffer A             24     μl                                             buffer B             6      μl                                             ______________________________________                                    

Incubate for 30-60 minutes at 16'C. For library preparation, use 5 timesthe volumes shown above. The ligation product is concentrated andpurified using Geneclean and eluted into a volume of 10-15 ul of water.This is introduced into electrocompetent TG1 cells where typicaltransformation efficiencies are as follows:

    ______________________________________                                        pUC119            1.6 × 10.sup.9 transformants/μg                    ligated vector with insert                                                                      1.0 × 10.sup.7 transformants/μg                    ligated vector without insert                                                                   2.0 × 10.sup.6 transformants/μg                    ______________________________________                                    

6. Selection of chain shuffled clones

After each chain shuffling procedure clones which bind to TNF-a areselected by the procedure described in 2 above for selection from themouse antibody library and binding assayed by ELISA.

7. Further shuffles

Following the initial shuffle e.g. of a selected population of mouseheavy chain variable domains with human naive light chain variabledomains, the selected clones from this first shuffle are used as asource of miniprep DNA to repeat the chain shuffling and cloningprocedure described in 4 and 5 above, e.g. with the human naive heavychain variable domains of the library derived in WO 92/01047, example42. Selection is then performed on this second shuffled library toisolate fully humanised antibodies binding to TNF-alpha derived by thisrepertoire imprinted selection procedure.

EXAMPLE 5 Creation of a Synthetic Library

By display of antibody repertoires on the: surface of filamentous phageand selection of the phage with antigen¹, we can mimic immuneselection².3 and make human antibodies from the rearranged V-genes ofunimmunised donors⁴. Human antibodies have now been made by synthesisfrom defined V-gene elements. A repertoire of 49 human germ line V_(H)gene segments was rearranged in vitro by joining to a synthetic"D-segment" of five random amino acid residues and a J-segment, tocreate a synthetic third complementarity determining region⁵ (CDR) ofeight residues. The rearranged V_(H) genes were cloned with a human Vλ3light chain as single-chain Fv fragments for phage display. The libraryof 10⁷ phages was panned with a hapten 2-phenyl-oxazol-5-one (phOx)conjugate to bovine serum albumin (BSA), and phage isolated that encodedfragments with specific binding activity to phOx-BSA, and withaffinities to phOx-gamma-aminobutyric acid (phOx-GABA) in the micromolarrange. Comparison of twenty one clones with unique sequences showed thatthe in vitro "immune response" to the hapten was largely restricted tothe V_(H) 26 segment (V_(H) 3 family)⁶ with an invariant aromaticresidue (Tyr, Phe, Trp) at residue 98 of CDR3. The use of V-genesrearranged in vitro may allow the design of antibody libraries biasedtowards the binding of antigens of known structure, and the creation oftherapeutic human antibodies with reduced immunogenicity.

Antibody variable domains consist of a β-sheet framework with threeloops of hypervariable sequence or CDRs⁵. The loops create antigenbinding sites of a variety of shapes, ranging from flat surfaces⁷ topockets⁸. For human heavy chains, the sequence diversity of the firsttwo CDRs are encoded by a repertoire of about fifty germ line V_(H)segments. (I. M. Tomlinson et al., supra). The third CDR is generatedfrom the recombination of these segments with about thirty D and six Jsegments⁹, and although its sequence is highly variable, it oftenincludes a salt bridge from Asp101 of the loop to Arg94 of theframework¹⁰. The structures and lengths of the first two CDRs arerestricted¹⁰,11, but those of CDR3 differ greatly, with lengths rangingfrom 4 to 25 residues⁵.

A library was created of rearranged V_(H) genes with a CDR3 of eightresidues including Asp101, in combination with a single Vλ (ref.12)light chain. Forty nine germ line V_(H) segments encoding most of thehuman V_(H) repertoire (Tomlinson et al., supra) were each amplifiedusing the polymerase chain reaction¹³ and oligonucleotide primers thatintroduce a synthetic D-segment (of 15 bases of random sequence at the3' end of the V_(H) segment) and a J-segment, together encoding a CDR3loop of eight residues (FIG. 14). The rearranged segments were pooledand cloned for phage display with a human Vλ3 light chain, creating asynthetic library of 10⁷ phage clones. Like the immune system, thesynthetic library of 10⁷ phage clones can tap only a small fraction ofthe potential diversity. Thus the diversity is potentially 49×32⁵=1.6×10⁹ different nucleotide sequences, or 49×20⁵ =1.6×10⁸ differentamino acid sequences.

The library was subjected to four rounds of growth and panning onphOx-bovine serum albumin (BSA) coated tubes, and clones screened assoluble¹⁴ single chain Fv fragments¹⁵,16 for binding activity tophOx-BSA by ELISA⁴. After the third and fourth rounds, 14/96 and 61/96clones respectively were identified with binding activities to phOx-BSAand of these (29 tested) none bound to other proteins (see legend Table4). Furthermore their binding to phOx-BSA coated plates could becompeted with the soluble hapten (Table 4).

Sequencing revealed that many (21/29) of the phOx binders were unique,with an eight residue CDR3, and utilised either a segment from the V_(H)4 family, or one of three segments from the V_(H) 3 family (Table 4).Together these segments use three of the seven "canonical" foldsavailable to the first two hypervariable loops of human V_(H) segments.(C. Chothia, et al., supra). The majority of the unique clones (16/21)were derived from the VH26 segment⁶ and have related sequences in thethird hypervariable loop: in this group the first residue tends to havea branched aliphatic side chain (15/16), the second residue tends to belysine or arginine (11/16), while the fourth residue is always anaromatic residue (most frequently tyrosine).

The affinities (K_(d)) of two of the stronger binders (Ox 13 and Ox-31,Table 4) for phOx-GABA were determined by fluorescence quenchtitration¹⁷ as 3.1±0.2 μM and 6.7±0.7 μM respectively. Although thesynthetic antibody library lacks the diverse VH-CDR3 lengths and thedifferent light chains of antibodies made in vivo, the affinities forphOx-GABA compare with 0.5 μM for a (phage) antibody made fromunimmunised human donors⁴, or 1 μM for several hybridomas from a mouseprimary immune response¹⁸ (but see caveat, Table 3 legend). To improvethese affinities, one could systematically alter (see below) the manydifferent phOx antibodies selected (Table 3).

In principle, the use of phage display libraries of V-genes rearrangedin vitro offers an attractive alternative to those rearranged in vivo⁴.Firstly the framework regions and first two hypervariable loops of bothheavy and light chains of the synthetic human antibodies created fromthe library are essentially germ line. This contrasts with the "primary"phage antibodies tapped from human V-genes rearranged in vivo, in whichthe extent of somatic mutation varied widely⁴. Leaving asidepolymorphism, the VH gene segments are identical in differentindividuals, and the synthetic antibodies are potentially lessimmunogenic. By altering the lengths and sequences of the heavy andlight chain CDR3 loops, or by localising the minimal mutations in theother CDR loops, or by shuffling with synthetic "germ line" lightchains¹⁹,20, it may be possible to improve their affinities whileretaining their germ line character.

Secondly both kinds of libraries are highly biased. In the "natural"libraries, the bias is outside our control, and is imposed for exampleby allelic variation, deletion polymorphism and deletion ofself-reactive clones. In the synthetic library, the bias can beintroduced systematically. Here for example, all the VH-gene segments,were chosen and thereby the folding of the first and secondhypervariable loops: also fixed were the length and diversity of VH-CDR3and the light chain. Although several ways of making diverse syntheticlibraries have been suggested², it should also be possible toincorporate design principles into the encoded structures. If the shapeof the antigen were known, an envelope of roughly complementary bindingsites might be designed and built with defined V-gene elements. Use ofsuch "designer" libraries would favour the isolation of antibodies withhigher affinities.

                  TABLE 3                                                         ______________________________________                                                                           Library                                          No. of                       size ×                               Family                                                                              genes   VH segments*         10.sup.-6 (%)                              ______________________________________                                        V.sub.H 1                                                                           14      1-5, 7, 8, 10, 12, 14, 15, 20, 21, 25                                                              2.3  (20)                                  V.sub.H 2                                                                           1       27                   1.0  (9)                                   V.sub.H 3                                                                           23      29-33, 35, 38-40, 42, 44-54, 58, 59                                                                2.1  (19)                                  V.sub.H 4                                                                           9       63-71                2.6  (23)                                  V.sub.H 5                                                                           1       73                   1.4  (12)                                  V.sub.H 6                                                                           1       74                   1.9  (17)                                  Total:                                                                              49                           11.3 (100)                                 ______________________________________                                         *for simplicity V.sub.H segments are listed according to DP nomenclature      of Tomlinson et al., supra.                                              

Table 3 Composition of the Synthetic Library

Forty nine human V_(H) segments (Tomlinson et al, supra) were used, onefor each of the V_(H) 2, V_(H) 5 and V_(H) 6 gene families and multiplesegments for the other three families, and cloned according to family.Clones from the V_(H) segments of each family (see legend FIG. 1) werechecked for presence of insert (on average 85%) and pooled into a singlelarge library as in Table 4, creating a (controlled) bias for certaingene families. The segments from the V_(H) 2, V_(H) 5, V_(H) 6 familiesare thereby "overrepresented" with respect to the segments from otherfamilies. Sequencing of thirty five clones from the unselected libraryconfirmed that V_(H) segments from each family were present, and thatthe nucleotides were present in the expected ratios in the D-segment,but with a slight bias for C. (At the first and second position of eachcodon, A, 21.3%; G, 17.9%; C33.7% and T, 27.1%; at the third position,G, 42.6% and T, 57.4%). The expression levels of the antibody fragmentswere also checked, and V_(H) segments were identified in clones withdetectable expression levels, for example V_(H) 1 (DP-7), V_(H) 2(DP-27), V_(H) 3 (DP-29,35,38,44,47,51,53), V_(H) 4 (DP-63,69), V_(H) 5(DP-73) and V_(H) 6 (DP-74).

Methods

The clones were checked for presence of insert by `PCR-screening`²¹ witholigonucleotides LMB3 and pHEN-SEQ (ref.4) and sequenced fromdouble-stranded DNA by the dideoxy chain termination method²² witholigonucleotide LINKSEQ (5'-CGA TCC GCC ACC GCC AGA G-3'). (The numbersin the tables are corrected for insert). Expression of soluble scFvfragments was checked by spotting 10 μl supernatant of induced overnightCultures in E. coli HB2151 (ref.14) onto a nitrocellulose filter using aslot-blot device (Minifold II, Schleicher and Schuell), and detectingthe bound peptide-tagged scFv fragments with 9E10 antibody²³ andperoxidase labelled anti-mouse antibodies (Sigma).

                  TABLE 4                                                         ______________________________________                                                        Germline   Canonical Loop                                     Clone   Family  gene*      structure*                                                                              I.sub.50.sup.φ                       ______________________________________                                        Ox-31   V.sub.H 3                                                                             DP-42      1-1        26                                      Ox-15   V.sub.H 3                                                                             DP-45      1-1       >300                                     Ox-18   "       "          "         >300                                     Ox-33   V.sub.H 3                                                                             DP-47      1-3        20                                      Ox-13   "       "          "          50                                      Ox-9    "       "          "          80                                      Ox-7    "       "          "          86                                      Ox-30   "       "          "          86                                      Ox-12   "       "          "          86                                      Ox-5    "       "          "         100                                      Ox-3    "       "          "         125                                      Ox-20   "       "          "         125                                      Ox-21   "       "          "         125                                      Ox-4    "       "          "         130                                      Ox-10   "       "          "         150                                      Ox-14   "       "          "         180                                      Ox-19   "       "          "         250                                      Ox-25   "       "          "         >400                                     Ox-27   "       "          "         ¶                              Ox-2§                                                                            V.sub.H 4                                                                             DP-67      2-1       >400                                     Ox-1    "       "          "         >400                                     ______________________________________                                         *Tomlinson et al., supra, Chothia et al., supra.                              .sup.φ in μM, according to competition ELISA with phOxGABA.            .sup.§ shows V67A mutation in FR3.                                       .sup.¶ Not determined.                                         

Table 4 phOx-Binders Isolated from the Synthetic Library

Phage were prepared from the library by rescue with VCS-M13, andsubjected to rounds of panning in phOx-BSA coated tubes as in ref.4. Thesequences of 21 phage binding to phOx revealed four germ line VHsegments, DP-42,45,47 (VH3 family) and DP-67 (VH4 family). DP-47 isidentical to VH26 (ref.6, corrected in ref.24), while DP-42, DP-45 andDP-67 only differ in one or a few framework residues from 8-1B (ref.25),65-2 (ref.26) or VH4.22 (ref.27) respectively. Clones from theunselected library using the DP47 V_(H) segment and lacking thecharacteristic pattern of CDR3 did not bind to phOx. Of the 21 phOxbinders tested, none bound to BSA, NIP-BSA, plastic, chymotrypsinogen A,cytochrome c, bovine thyroglobulin, keyhole limpet haemocyanin or turkeyegg white lysozyme. Four clones that bound to BSA (but not to phOx) werefound to be contaminants (αBSA3 clones, from ref.4).

Methods

As in ref.4. The relative affinities of the scFv fragments weredetermined by inhibition ELISA²⁸. A serial dilution of4-gamma-amino-butyric acid methylene12-phenyloxazol-5-one (phOx-GABA),with concentrations ranging from 6 to 400 μM, was made in 4% Marvel-PBS,and scFv supernatant added. The concentration of phOx-GABA resulting ina 50% reduction of the signal (I₅₀) for binding to phOx-BSA was noted.The affinities of the clones Ox-13 and Ox-31 for phOx-GABA weredetermined by fluorescence quench titration using scFv purified by thec-myc tag (ref.4). Ideally, the affinity for the phOx-BSA conjugatewould have been measured directly, or that for phOx-caproic acid, butphOx-GABA was used here to allow comparison with the hybridoma data ofref.18. The affinities of the antibodies for the phOx conjugate, or forphOx-caproic acid are likely to be better than those measured forphOx-GABA.

FIG. 14 Assembly of Rearranged VH Genes (See Text) Methods

A synthetic oligonucleotide SYNLIB15' GCC TCC ACC TCT CGA GAC GGT GACCAG GGT ACC TTG GCC CCA ATA GTC AAA ([A/C]NN)₅ TCT TGC ACA GTA ATA CACGGC CGT GTC 3' (SEQ ID NO: 109, see Table 1) introduced a D-segment witha five residue random amino acid sequence, a J-segment and an XhoIrestriction site, to the 3' end of each of 49 human V_(H) germlinesegments (Tomlinson et al., supra). The primer was used in thepolymerase chain reaction¹³ with a V_(H) family based back primer(VHBACK) incorporating an NcoI site⁴. Each V_(H) segment clone (providedas single stranded template in M13 vector) was amplified separately at94° C. for 1 min, 50° C. for 1 min, and 72° C. for 1.5 min, for 25cycles, on a PHC-3 thermocycler (Techne). Each amplification was checkedby electrophoresis on agarose gel, and similar amounts of DNA from V_(H)segments of the same family were pooled, digested with NcoI and XhoI,and cloned into the vector pHEN1 (ref.14) carrying a rearranged Vλ3light chain variable domain (IGLV3S1; ref.12) taken from a scFv fragmentbinding to BSA⁴.

If, instead of a random oligonucleotide, an oligonucleotide encoding aCDR, eg rodent, is used, this would imprint that non-human CDR on theproduct synthetic human library.

References Mentioned in Example 5

1. McCafferty, J., Griffiths, A. D., Winter, G. & Chiswell D. J. (1990).Nature, 348, 552-554.

2. Milstein, C. (1990). Proc R Soc Lond Biol, 239, 1-16.

3. Winter, G. & Milstein, C (1991). Nature, 349, 293-299.

4. Marks, J. D., et al (1991). J Mol Biol, 222, 581-597.

5. Kabat, E. A., Wu, T. T., Reid-Miller, M., Perry, H. M. & Gottesman,K. S. Sequences of proteins of immunological interest (U.S. Departmentof Health and Human Services, U.S. Government Printing Office, 1987).

6. Matthyssens, G. & Rabbits, T. H. (1980). Proc Natl Acad Sci U.S.A.,77, 6561-6565.

7. Amir, A. G., Mariuzza, R. A., Phillips, S. E. & Poljak, R. J. (1986).Science, 233, 747-753.

8.

8. Alzari, P. M., et al (1990). Embo J, 9, 3807-3814.

9. Ichihara, Y., Matsuoka, H. & Kurosawa, Y (1988). Embo J, 7,4141-4150.

10. Chothia, C. & Lesk, A. M. (1987). J Mol Biol, 196, 901-917.

11. Chothia, C., et al (1989). Nature, 342, 877-883.

12. Frippiat, J. P., et al (1990). Nucleic Acids Res, 18, 7134.

13. Saiki, R. K., et al (1985). Science, 230, 1350-1354.

14. Hoogenboom, H. R., et al (1991). Nucleic Acids Res, 19, 4133-4137.

15. Huston, J. S., et al (1988). Proc Natl Acad Sci U.S.A., 85,5879-5883.

16. Bird, R. E., et al (1988). Science, 242, 423-426.

17. Eisen, H. N. (1964). Meth Med Research, 10, 115-121.

18. Foote, J. & Milstein, C. (1991). Nature, 352, 530-532.

19. Clackson, T., Hoogenboom, H. R., Griffiths, A. D. & Winter, G(1991). Nature, 352, 624-628.

20. Roberts, A. J., et al (1992). Bio/Technology, in press.

21. Gussow, D. & Clackson, T. (1989). Nucleic Acids Res, 17, 4000.

22. Sanger, F., Nicklen, S. & Coulson, A. R. (1977). Proc Natl Acad SciU.S.A., 74, 5463-5467.

23. Munro, S. & Pelham, H. R. B. (1986). Cell, 46, 291-300.

24. Chen, P. P., Liu, M. F., Sinha, S. & Carson, D. A. (1988). ArthritisRheum, 31, 1429-1431.

25. Berman, J. E., et al (1988). Embo J, 7, 727-738.

26. Matsuda, F., et al (1990). Embo J, 9, 2501-2506.

27. Sanz, I., et al (1989). Embo J, 8, 3741-3748.

28. Rath, S., Stanley, C. M. & Steward, M. W. (1988). J Immunol Methods,106, 245-249.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 144                                                (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 120 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GlnValLysLeuGlnGlnSerGlyAlaGluLeuValLysProGlyAla                              151015                                                                        SerValLysMetSerCysLysAlaSerGlyTyrThrPheAlaSerTyr                              202530                                                                        TrpIleAsnTrpValLysGlnArgProGlyGlnGlyLeuGluTrpIle                              354045                                                                        GlyHisIleTyrProValArgSerIleThrLysTyrAsnGluLysPhe                              505560                                                                        LysSerLysAlaThrLeuThrLeuAspThrSerSerSerThrAlaTyr                              65707580                                                                      MetGlnLeuSerSerLeuThrSerGluAspSerAlaValTyrTyrCys                              859095                                                                        SerArgGlyAspGlySerAspTyrTyrAlaMetAspTyrTrpGlyGln                              100105110                                                                     GlyThrThrValThrValSerSer                                                      115120                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 360 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CAGGTCAAACTGCAGCAGTCAGGGGCTGAGCTTGTGAAGCCTGGGGCTTCAGTGAAAATG60                TCCTGCAAGGCTTCTGGCTATACCTTCGCCAGCTACTGGATAAACTGGGTGAAGCAGAGG120               CCTGGACAAGGCCTTGAGTGGATTGGACATATTTATCCTGTTAGAAGTATTACTAAGTAC180               AATGAGAAGTTCAAGAGTAAGGCCACACTGACTCTAGACACATCCTCCAGCACAGCCTAC240               ATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTATTGTTCAAGAGGGGAC300               GGCAGTGATTATTATGCTATGGACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA360               (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 107 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AspIleGluLeuThrGlnSerProAlaIleLeuSerAlaSerProGly                              151015                                                                        GlyLysValThrMetThrCysArgAlaSerSerSerValSerTyrMet                              202530                                                                        HisTrpTyrGlnGlnLysProGlySerSerProLysProTrpIleTyr                              354045                                                                        AlaThrSerAsnLeuAlaSerGlyValProThrArgPheSerGlyThr                              505560                                                                        GlySerGlyThrSerTyrSerLeuThrIleSerArgValGluAlaGlu                              65707580                                                                      AspAlaAlaThrTyrTyrCysGlnGlnTrpSerArgAsnProPheThr                              859095                                                                        PheGlySerGlyThrLysLeuGluIleLysArg                                             100105                                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 321 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GACATTGAGCTCACCCAGTCTCCAGCAATCCTGTCTGCATCTCCAGGGGGGAAGGTCACA60                ATGACTTGTAGGGCCAGCTCAAGTGTAAGTTACATGCACTGGTACCAGCAGAAGCCAGGA120               TCCTCCCCCAAACCCTGGATTTATGCCACATCCAACCTGGCTTCTGGAGTCCCTACTCGC180               TTCAGTGGCACTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGGGTGGAGGCTGAA240               GATGCTGCCACTTATTACTGCCAGCAGTGGAGTCGTAACCCATTCACGTTCGGCTCGGGC300               ACCAAGCTGGAAATCAAACGG321                                                      (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CCGTTTGATTTCCAGCTTGGTGCC24                                                    (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 54 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CATGACCACGCGGCCCAGCCGGCCATGGCCGACATTGAGCTCACCCAGTCTCCA54                      (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 48 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GGCACCAAGCTGGAAATCAAACGGACTGTGGCTGCACCATCTGTCTTC48                            (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 54 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GAGTCATTCTCGACTTGCGGCCGCTTATTAACACTCTCCCCTGTTGAAGCTCTT54                      (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       TGAGGAGACGGTGACCGTGGTCCCTTGGCCCC32                                            (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 51 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GGGACCACGGTCACCGTCTCCTCAGGAAGTGCATCCGCCCCAACCCTTTTC51                         (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      CCACGATTCTGCGGCCGCCACTGGAAGAGGCACGTTCTTTTCTTT45                               (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 48 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GAGTCATTCTCGACTTGCGGCCGCACACTCTCCCCTGTTGAAGCTCTT48                            (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 51 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GAGTCATTCTCGACTTGCGGCCGCTGAACATTCTGTAGGGGCCACTGTCTT51                         (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      CACAGTGCACTCGACATTGAGCTCACCCAGTCTCCA36                                        (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      CCACGATTCTGCGGCCGCCACTGGAAGAGGCACGTTCTTTTCTTT45                               (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      TGGAAGAGGCACGTTCTTTTCTTT24                                                    (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      TGAACATTCTGTAGGGGCCACTGTCTT27                                                 (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      ACACTCTCCCCTGTTGAAGCTCTT24                                                    (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      GCGATGGTTGTTGTCATTGTCGGC24                                                    (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      GAATTTTCTGTATGAGG17                                                           (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      GTGTGGCCTTGTTGGCTTG19                                                         (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      CTATGCGGCCCCATTCA17                                                           (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      CGATCCGCCACCGCCAGAG19                                                         (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      AGGAAGTCCTGTGCGAGGCAG21                                                       (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 56 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      GTCCTCGCAACTCGCGCCCAGCCGGCCATGGCCCAGTCTGTGTTGACGCAGCCGCC56                    (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      CCACGATTCTGCGGCCGCCTATGAACATTCTGTAGGGGTCACTGT45                               (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      GCCTGAACCGCCTCCACCTCTCGAGAACGGTGACCAGGG39                                     (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      CTGGTCACCGTCTCGAGAGGTGGAGGC27                                                 (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      GGAGGATGCACTTGTCGACACGGTGACCAG30                                              (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      CAGGTGCAGCTGGTGCAGTCTGG23                                                     (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      CAGGTCAACTTAAGGGAGTCTGG23                                                     (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      GAGGTGCAGCTGGTGGAGTCTGG23                                                     (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      CAGGTGCAGCTGCAGGAGTCGGG23                                                     (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      GAGGTGCAGCTGTTGCAGTCTGC23                                                     (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      CAGTGACAGCTGCAGCAGTCAGG23                                                     (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      TGGAAGAGGCACGTTCTTTTCTTT24                                                    (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      AGACTCTCCCCTGTTGAAGCTCTT24                                                    (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      TGAAGATTCTGTAGGGGCCACTGTCTT27                                                 (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      TGAGGAGACGGTGACCAGGGTGCC24                                                    (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      TGAAGAGACGGTGACCATTGTCCC24                                                    (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      TGAGGAGACGGTGACCAGGGTTCC24                                                    (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      TGAGGAGACGGTGACCGTGGTCCC24                                                    (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      CATGACCACAGTGCACAGGTGCAGCTGGTGCAGTCTGG38                                      (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      CATGACCACAGTGCACAGGTCAACTTAAGGGAGTCTGG38                                      (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      CATGACCACAGTGCACAGGTGCAGCTGGTGGAGTCTGG38                                      (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      CATGACCACAGTGCACAGGTGCAGCTGCAGGAGTCGGG38                                      (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      CATGACCACAGTGCACAGGTGCAGCTGTTGCAGTCTGC38                                      (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      CATGACCACAGTGCACAGGTACAGCTGCAGCAGTCAGG38                                      (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      GAGTCATTCTCGTGTCGACACGGTGACCAGGGTGCC36                                        (2) INFORMATION FOR SEQ ID NO:50:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                      GAGTCATTCTCGTGTCGACACGGTGACCATTGTCCC36                                        (2) INFORMATION FOR SEQ ID NO:51:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                      GAGTCATTCTCGTGTCGACACGGTGACCAGGGTTCC36                                        (2) INFORMATION FOR SEQ ID NO:52:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                      GAGTCATTCTCGTGTCGACACGGTGACCGTGGTCCC36                                        (2) INFORMATION FOR SEQ ID NO:53:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 56 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                      GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGTCTGTGTTGACGCAGCCGCC56                    (2) INFORMATION FOR SEQ ID NO:54:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 56 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                      GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGTCTGCCCTGACTCAGCCTGC56                    (2) INFORMATION FOR SEQ ID NO:55:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 56 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                      GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCTCCTATGTGCTGACTCAGCCACC56                    (2) INFORMATION FOR SEQ ID NO:56:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 56 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                      GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCTCTTCTGAGCTGACTCAGGACCC56                    (2) INFORMATION FOR SEQ ID NO:57:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 56 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                      GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGGTTATACTGACTCAACCGCC56                    (2) INFORMATION FOR SEQ ID NO:58:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 56 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                      GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGGCTGTGCTCACTCAGCCGTC56                    (2) INFORMATION FOR SEQ ID NO:59:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 56 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                      GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCAATTTTATGCTGACTCAGCCCCA56                    (2) INFORMATION FOR SEQ ID NO:60:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 56 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                                      GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGACATCCAGATGACCCAGTCTCC56                    (2) INFORMATION FOR SEQ ID NO:61:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 56 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                                      GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGATGTTGTGATGACTCAGTCTCC56                    (2) INFORMATION FOR SEQ ID NO:62:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 56 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                                      GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGAAATTGTGTTGACGCAGTCTCC56                    (2) INFORMATION FOR SEQ ID NO:63:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 56 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                                      GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGACATCGTGATGACCCAGTCTCC56                    (2) INFORMATION FOR SEQ ID NO:64:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 56 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                                      GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGGAACGACACTCACGCAGTCTCC56                    (2) INFORMATION FOR SEQ ID NO:65:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 56 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                                      GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGAAATTGTGCTGACTCAGTCTCC56                    (2) INFORMATION FOR SEQ ID NO:66:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 78 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                                      GAGTCATTCTCGACTTGCGGCCGCCTGCTATTATCGGGCGCGCCTTTATTATGAAGATTC60                TGTAGGGGCCACTGTCTT78                                                          (2) INFORMATION FOR SEQ ID NO:67:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 75 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                                      GAGTCATTCTCGACTTGCGGCCGCCTGCTATTATCGGGCGCGCCTTTATTAAGACTCTCC60                CCTGTTGAAGCTCTT75                                                             (2) INFORMATION FOR SEQ ID NO:68:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                                      AGACTCTCCCCTTTTGAAGCTCTT24                                                    (2) INFORMATION FOR SEQ ID NO:69:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:                                      TGAAGATTCTGTAGGGGCCACTGTCTT27                                                 (2) INFORMATION FOR SEQ ID NO:70:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 48 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:                                      GAGTCATTCTCGACTTGCGGCGGCAGACTCTCCCCTGTTGAAGCTCTT48                            (2) INFORMATION FOR SEQ ID NO:71:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 51 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:                                      GAGTCATTCTCGACTTGCGGCCGCTGAAGATTCTGTAGGGGCCACTGTCTT51                         (2) INFORMATION FOR SEQ ID NO:72:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:                                      GACATCCAGATGACCCAGTCTCC23                                                     (2) INFORMATION FOR SEQ ID NO:73:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:                                      GATGTTGTGATGACTCAGTCTCC23                                                     (2) INFORMATION FOR SEQ ID NO:74:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:                                      GAAATTGTGTTGACGCAGTCTCC23                                                     (2) INFORMATION FOR SEQ ID NO:75:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:                                      GACATCGTGATGACCCAGTCTCC23                                                     (2) INFORMATION FOR SEQ ID NO:76:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:                                      GAAACGACACTCACGCAGTCTCC23                                                     (2) INFORMATION FOR SEQ ID NO:77:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:                                      GAAATTGTGCTGACTCAGTCTCC23                                                     (2) INFORMATION FOR SEQ ID NO:78:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:                                      CAGTCTGTGTTGACGCAGCCGCC23                                                     (2) INFORMATION FOR SEQ ID NO:79:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:                                      CAGTCTGCCCTGACTCAGCCTGC23                                                     (2) INFORMATION FOR SEQ ID NO:80:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:                                      TCCTATGTGCTGACTCAGCCACC23                                                     (2) INFORMATION FOR SEQ ID NO:81:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:                                      TCTTCTGAGCTGACTCAGGACCC23                                                     (2) INFORMATION FOR SEQ ID NO:82:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:                                      CACGTTATACTGACTCAACCGCC23                                                     (2) INFORMATION FOR SEQ ID NO:83:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:                                      CAGGCTGTGCTCACTCAGCCGTC23                                                     (2) INFORMATION FOR SEQ ID NO:84:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:                                      AATTTTATGCTGACTCAGCCCCA23                                                     (2) INFORMATION FOR SEQ ID NO:85:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 72 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:                                      GTCCTCGCAACTGGCGCGCCACAATTTCACAGTAAGGAGGTTTAACTTGTGAAAAAATTA60                TTATTCGCAATT72                                                                (2) INFORMATION FOR SEQ ID NO:86:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 108 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:                                      GGATGCACTTGTCGACACGGTGACCAGGGTACCTTGGCCCCAGTAGTCAAAGTAGTAGTC60                CTCTTCGTAATCATAGTAGATCAGGTCACAGTAATACACGGCCGTGTC108                           (2) INFORMATION FOR SEQ ID NO:87:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 108 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:                                      GGATGCACTTGTCGACACGGTGACCAGGGTACCTTGGCCCCAGTAGTCAAAGTAGTAGTC60                CTCTTCGTAATCATAGTAGATCAGGTCACAGTAATACACGGCCGTGTC108                           (2) INFORMATION FOR SEQ ID NO:88:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:                                      TGAGCACACAGTGCACTCCAGTCTGTGTTGACGCAGCCGCC41                                   (2) INFORMATION FOR SEQ ID NO:89:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:                                      TGAGCACACAGTGCACTCCAGTCTGCCCTGACTCAGCCTGC41                                   (2) INFORMATION FOR SEQ ID NO:90:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:                                      TGAGCACACAGTGCACTCTCCTATGTGCTGACTCAGCCACC41                                   (2) INFORMATION FOR SEQ ID NO:91:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:                                      TGAGCACACAGTGCACTCTCTTCTGAGCTGACTCAGGACCC41                                   (2) INFORMATION FOR SEQ ID NO:92:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:                                      TGAGCACACAGTGCACTCCAGGTTATACTGACTCAACCGCC41                                   (2) INFORMATION FOR SEQ ID NO:93:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:                                      TGAGCACACAGTGCACTCCAGGCTGTGCTCACTCAGCCGTC41                                   (2) INFORMATION FOR SEQ ID NO:94:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:                                      TGAGCACACAGTGCACTCAATTTTATGCTGACTCAGCCCCA41                                   (2) INFORMATION FOR SEQ ID NO:95:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:                                      TGAGCACACAGTGCACTCGACATCCAGATGACCCAGTCTCC41                                   (2) INFORMATION FOR SEQ ID NO:96:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:                                      TGAGCACACAGTGCACTCGATGTTGTGATGACTCAGTCTCC41                                   (2) INFORMATION FOR SEQ ID NO:97:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:                                      TGAGCACACAGTGCACTCGAAATTGTGTTGACGCAGTCTCC41                                   (2) INFORMATION FOR SEQ ID NO:98:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:                                      TGAGCACACAGTGCACTCGACATCGTGATGACCCAGTCTCC41                                   (2) INFORMATION FOR SEQ ID NO:99:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:99:                                      TGAGCACACAGTGCACTCGAAACGACACTCACGCAGTCTCC41                                   (2) INFORMATION FOR SEQ ID NO:100:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:100:                                     TGAGCACACAGTGCACTCGAAATTGTGCTGACTCAGTCTCC41                                   (2) INFORMATION FOR SEQ ID NO:101:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 56 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:101:                                     GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGGTGCAGTCTGG56                    (2) INFORMATION FOR SEQ ID NO:102:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 56 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:102:                                     GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGGTCAACTTAAGGGAGTCTGG56                    (2) INFORMATION FOR SEQ ID NO:103:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 56 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:103:                                     GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGGTGGAGTCTGG56                    (2) INFORMATION FOR SEQ ID NO:104:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 56 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:104:                                     GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGCAGGAGTCGGG56                    (2) INFORMATION FOR SEQ ID NO:105:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 56 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:105:                                     GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGTTGCAGTCTGC56                    (2) INFORMATION FOR SEQ ID NO:106:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 56 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:106:                                     GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGGTACAGCTGCAGCAGTCAGG56                    (2) INFORMATION FOR SEQ ID NO:107:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:107:                                     CATGACCACAGTGCACAGGTSMARCTGCAGSAGTCWGG38                                      (2) INFORMATION FOR SEQ ID NO:108:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 57 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:108:                                     CATGCCATGACTCGCGGCCCAGCCGGCCATGGCCSAGGTSMARCTGCAGSAGTCWGG57                   (2) INFORMATION FOR SEQ ID NO:109:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 93 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:109:                                     GCCTCCACCTCTCGAGACGGTGACCAGGGTACCTTGGCCCCAATAGTCAAAMNNMNNMNN60                MNNMNNTCTTGCACAGTAATACACGGCCGTGTC93                                           (2) INFORMATION FOR SEQ ID NO:110:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 105 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:110:                                     AlaIleGluLeuThrGlnProAlaIleLeuSerAlaSerProGlyGly                              151015                                                                        LysValThrMetThrCysArgAlaSerSerSerValSerTyrMetHis                              202530                                                                        TrpTyrGlnGlnLysProGlySerSerProLysProTrpIleTyrAla                              354045                                                                        ThrSerAsnLeuAlaSerGlyValProThrArgPheSerGlySerGly                              505560                                                                        ThrGlyThrSerTyrSerLeuThrIleSerArgValGluAlaGluAsp                              65707580                                                                      AlaAlaThrTyrTyrCysGlnGlnTrpSerArgAsnProPheThrPhe                              859095                                                                        GlySerGlyThrLysLeuGluIleLys                                                   100105                                                                        (2) INFORMATION FOR SEQ ID NO:111:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 98 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:111:                                     GlnSerValLeuThrGlnProProSerAlaSerGlyThrProGlyGln                              151015                                                                        ArgValThrIleSerCysSerGlySerSerSerAsnIleGlySerAsn                              202530                                                                        TyrValTyrTrpTyrGlnGlnLeuProGlyThrAlaProLysLeuLeu                              354045                                                                        IleTyrArgAsnAsnGlnArgProSerGlyValProAspArgPheSer                              505560                                                                        GlySerLysSerGlyThrSerAlaSerLeuAlaIleSerGlyLeuArg                              65707580                                                                      SerGluAspGluAlaAspTyrTyrCysAlaAlaTrpAspAspSerLeu                              859095                                                                        SerGly                                                                        (2) INFORMATION FOR SEQ ID NO:112:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 113 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:112:                                     GlnSerValLeuThrGlnProSerSerValSerAlaAlaProGlyGln                              151015                                                                        LysValThrIleSerCysSerGlySerSerSerAsnIleGlyAsnAsn                              202530                                                                        TyrValTyrTrpTyrGlnGlnLeuProGlyThrAlaProLysLeuLeu                              354045                                                                        IleTyrArgAsnAsnGlnArgProSerGlyValProAspArgPheSer                              505560                                                                        GlySerLysSerGlySerSerAlaSerLeuAlaIleSerGlyLeuArg                              65707580                                                                      SerGluAspGluAlaAspTyrTyrCysAlaAlaTrpAspAspSerLeu                              859095                                                                        SerGlyArgArgValValPheGlyGlyGlyThrLysLeuThrValLeu                              100105110                                                                     Gly                                                                           (2) INFORMATION FOR SEQ ID NO:113:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 113 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:113:                                     GlnSerValLeuThrGlnProSerSerAlaSerGlyThrProGlyGln                              151015                                                                        ArgValThrIleSerCysSerGlySerSerSerAsnIleGlySerAsn                              202530                                                                        TyrValTyrTrpTyrGlnGlnLeuProGlyThrAlaProLysLeuLeu                              354045                                                                        IleTyrArgAsnAsnGlnArgProSerGlyValProAspArgPheSer                              505560                                                                        GlySerLysSerGlyThrSerAlaSerLeuAlaIleSerGlyLeuArg                              65707580                                                                      SerGluAspGluAlaAspTyrTyrCysAlaAlaTrpAspAspSerLeu                              859095                                                                        SerGlyArgAspValValPheGlyGlyGlyThrLysLeuThrValLeu                              100105110                                                                     Gly                                                                           (2) INFORMATION FOR SEQ ID NO:114:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 113 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:114:                                     GlnSerValLeuThrGlnProAlaSerAlaSerGlyThrProGlyGln                              151015                                                                        ArgValThrIleSerCysSerGlySerSerSerAsnIleGlySerAsn                              202530                                                                        TyrValTyrTrpTyrGlnGlnLeuProArgThrAlaProLysLeuLeu                              354045                                                                        IleTyrArgAsnAsnGlnArgProSerGlyValProAspArgPheSer                              505560                                                                        GlySerLysSerGlyThrSerAlaSerLeuAlaIleSerGlyLeuArg                              65707580                                                                      SerGluAspGluAlaAspTyrTyrCysAlaAlaTrpAspAspSerLeu                              859095                                                                        SerGlyArgValTyrValPheGlyThrGlyThrLysValThrValLeu                              100105110                                                                     Gly                                                                           (2) INFORMATION FOR SEQ ID NO:115:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 113 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:115:                                     GlnValLysLeuGlnGlnSerGlyAlaGluLeuValLysProGlyAla                              151015                                                                        SerValLysMetSerCysLysAlaSerGlyTyrThrPheAlaSerTyr                              202530                                                                        TrpIleAsnTrpValLysGlnArgProGlyGlnGlyLeuGluTrpIle                              354045                                                                        GlyHisIleTyrProValArgSerIleThrLysTyrAsnGluLysPhe                              505560                                                                        LysSerLysAlaThrLeuThrLeuAspThrSerSerSerThrAlaTyr                              65707580                                                                      MetGlnLeuSerSerLeuThrSerGluAspSerAlaValTyrTyrCys                              859095                                                                        SerArgGlyAspGlySerAspTyrTyrAlaMetAspTyrTrpGlyGln                              100105110                                                                     Gly                                                                           (2) INFORMATION FOR SEQ ID NO:116:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 98 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:116:                                     GluValGlnLeuValGluSerGlyGlyGlyLeuValGlnProGlyGly                              151015                                                                        SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerSerTyr                              202530                                                                        SerMetAsnTrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal                              354045                                                                        SerTyrIleSerSerSerSerSerThrIleTyrTyrAlaAspSerVal                              505560                                                                        LysGlyArgPheThrIleSerArgAspAsnAlaLysAsnSerLeuTyr                              65707580                                                                      LeuGlnMetAsnSerLeuArgAspGluAspThrAlaValTyrTyrCys                              859095                                                                        AlaArg                                                                        (2) INFORMATION FOR SEQ ID NO:117:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 110 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:117:                                     GlnValGlnLeuLeuGlnSerGlyGlyGlyLeuValGlnProGlyGly                              151015                                                                        SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerSerTyr                              202530                                                                        AlaMetSerTrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal                              354045                                                                        SerTyrIleSerSerSerSerSerThrIleTyrTyrAlaAspSerVal                              505560                                                                        LysGlyArgPheThrIleSerArgAspAsnAlaLysAsnThrLeuTyr                              65707580                                                                      LeuGlnMetAsnSerLeuArgAspGluAspThrAlaValTyrTyrCys                              859095                                                                        AlaArgSerLeuValGlyAlaLeuAspTyrTrpGlyGlnGly                                    100105110                                                                     (2) INFORMATION FOR SEQ ID NO:118:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 98 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:118:                                     GlnValGlnLeuValGluSerGlyGlyGlyValValGlnProGlyArg                              151015                                                                        SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerSerTyr                              202530                                                                        AlaMetHisTrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal                              354045                                                                        AlaValIleSerTyrAspGlySerAsnLysTyrTyrAlaAspSerVal                              505560                                                                        LysGlyArgPheThrIleSerArgAspAsnSerLysAsnThrLeuTyr                              65707580                                                                      LeuGlnMetAsnSerLeuArgAlaGluAspThrAlaValTyrTyrCys                              859095                                                                        AlaArg                                                                        (2) INFORMATION FOR SEQ ID NO:119:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:119:                                     ValArgSerSerSerArgThrProSerAspLysProValAlaHisVal                              151015                                                                        ValAla                                                                        (2) INFORMATION FOR SEQ ID NO:120:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:120:                                     CysThrArgProAsnAsnAsnThrArgArgSerIleArgIleGlnArg                              151015                                                                        GlyProGlyArgAlaPheValThrIleGlyLysIleGlyAsnMetArg                              202530                                                                        GlnAlaHisCysAsn                                                               35                                                                            (2) INFORMATION FOR SEQ ID NO:121:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:121:                                     TyrCysThrArgProAsnAsnAsnThrArgArgSerIleArgIleGln                              151015                                                                        ArgGlyProGlyArgAlaPheValThrIleGlyLysIleGlyAsnMet                              202530                                                                        ArgGlnAlaHisCysTyr                                                            35                                                                            (2) INFORMATION FOR SEQ ID NO:122:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:122:                                     CAGGAAACAGCTATGAC17                                                           (2) INFORMATION FOR SEQ ID NO:123:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:123:                                     GTCGTCTTTCCAGACGTTAGT21                                                       (2) INFORMATION FOR SEQ ID NO:124:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:124:                                     ACCGCCAGAGCCACCTCCGCC21                                                       (2) INFORMATION FOR SEQ ID NO:125:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:125:                                     GGCGGAGGTGGCTCTGGCGGT21                                                       (2) INFORMATION FOR SEQ ID NO:126:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 189 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 34..177                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: sig.sub.-- peptide                                              (B) LOCATION: 34..99                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 100..177                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:126:                                     GCATGCAAATTCTATTTCAAGGAGACAGTCATAATGAAATACCTATTGCCTACG54                      MetLysTyrLeuLeuProThr                                                         22-20                                                                         GCAGCCGCTGGATTGTTATTACTCGCGGCCCAGCCGGCCATGGCCCAG102                           AlaAlaAlaGlyLeuLeuLeuLeuAlaAlaGlnProAlaMetAlaGln                              15-10-51                                                                      GTGCAGCTGCAGGTCGACCTCGAGATCAAACGGGCGGCCGCAGAACAA150                           ValGlnLeuGlnValAspLeuGluIleLysArgAlaAlaAlaGluGln                              51015                                                                         AAACTCATCTCAGAAGAGGATCTGAATTAATAAGAATTC189                                    LysLeuIleSerGluGluAspLeuAsn                                                   2025                                                                          (2) INFORMATION FOR SEQ ID NO:127:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 97 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:127:                                     GlnValGlnLeuGlnGlnSerGlyAlaGluLeuAlaSerProGlyAla                              151015                                                                        SerValThrLeuSerCysLysAlaSerGlyTyrThrPheThrAspHis                              202530                                                                        IleMetAsnTrpValLysLysArgProGlyGlnGlyLeuGluTrpIle                              354045                                                                        GlyArgIlePheProValSerGlyGluThrAsnTyrAsnGlnPheMet                              505560                                                                        GlyLysAlaArgPheSerValAspArgSerSerSerThrValSerMet                              65707580                                                                      ValLeuAsnSerLeuThrSerGluAspProAlaValTyrTyrCysAsp                              859095                                                                        Leu                                                                           (2) INFORMATION FOR SEQ ID NO:128:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 101 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:128:                                     GlnValGlnLeuGlnGlnSerGlyProGlyLeuValLysProSerGln                              151015                                                                        ThrLeuSerLeuThrCysAlaIleSerGlyAspSerValSerSerAsn                              202530                                                                        SerAlaAlaTrpAsnTrpIleArgGlnSerProSerArgGlyLeuGlu                              354045                                                                        TrpLeuGlyArgThrTyrTyrArgSerLysTrpTyrAsnAspTyrAla                              505560                                                                        ValSerValLysSerArgIleThrIleAsnProAspThrSerLysAsn                              65707580                                                                      GlnPheSerLeuGlnLeuAsnSerValThrProGluAspThrAlaVal                              859095                                                                        TyrTyrCysAlaArg                                                               100                                                                           (2) INFORMATION FOR SEQ ID NO:129:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 97 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:129:                                     GlnValGlnLeuGlnGlnSerGlyAlaGluLeuAlaSerProGlyAla                              151015                                                                        SerValThrLeuSerCysLysAlaSerGlyTyrThrPheThrAspHis                              202530                                                                        IleMetAsnTrpValLysLysArgProGlyGlnGlyLeuGluTrpIle                              354045                                                                        GlyArgIlePheProValSerGlyGluThrAsnTyrAsnGlnPheMet                              505560                                                                        GlyLysAlaArgPheSerValAspArgSerSerSerThrValSerMet                              65707580                                                                      ValLeuAsnSerLeuThrSerGluAspProAlaValTyrTyrCysAsp                              859095                                                                        Leu                                                                           (2) INFORMATION FOR SEQ ID NO:130:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 98 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:130:                                     GlnMetGlnLeuValGlnSerGlyProGluValLysLysProGlyThr                              151015                                                                        SerValLysValSerCysLysAlaSerGlyPheThrPheThrSerSer                              202530                                                                        AlaValGlnTrpValArgGlnAlaArgGlyGlnArgLeuGluTrpIle                              354045                                                                        GlyTrpIleValValGlySerGlyAsnThrAsnTyrAlaGlnLysPhe                              505560                                                                        GlnGluArgValThrIleThrArgAspMetSerThrSerThrAlaTyr                              65707580                                                                      MetGluLeuSerSerLeuArgSerGluAspThrAlaValTyrTyrCys                              859095                                                                        AlaAla                                                                        (2) INFORMATION FOR SEQ ID NO:131:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 126 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..126                                                          (D) OTHER INFORMATION: /transl.sub.-- except=(pos: 115 .. 117,                aa: Glu)                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: sig.sub.-- peptide                                              (B) LOCATION: 1..24                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 25..126                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 115..117                                                        (D) OTHER INFORMATION: /label=amber                                           /note="Suppressible translational stop codon."                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:131:                                     GTTGTTCCTTTCTATTCTCAGAGTGCACAGGTCCAACTGCAGGTCGAC48                            ValValProPheTyrSerGlnSerAlaGlnValGlnLeuGlnValAsp                              8-515                                                                         CTCGAGATCAAACGGGCGGCCGCAGAACAAAAACTCATCTCAGAAGAG96                            LeuGluIleLysArgAlaAlaAlaGluGlnLysLeuIleSerGluGlu                              101520                                                                        GATCTGAATGGGGCCGCATAGACTGTTGAA126                                             AspLeuAsnGlyAlaAlaGluThrValGlu                                                2530                                                                          (2) INFORMATION FOR SEQ ID NO:132:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 162 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..162                                                          (D) OTHER INFORMATION: /transl.sub.-- except=(pos: 151 .. 153,                aa: Glu)                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: sig.sub.-- peptide                                              (B) LOCATION: 1..63                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 64..162                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 151..153                                                        (D) OTHER INFORMATION: /note="Suppressible translational                      stop codon."                                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:132:                                     GTGAAAAAATTATTATTCGCAATTCCTTTAGTTGTTTTCTATGCGGCC48                            ValLysLysLeuLeuPheAlaIleProLeuValValPheTyrAlaAla                              21-20-15-10                                                                   CAGCCGGCCATGGCCCAGGTCCAACTGCAGGTCGACCTCGAGATCAAA96                            GlnProAlaMetAlaGlnValGlnLeuGlnValAspLeuGluIleLys                              51510                                                                         CGGGCGGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGGG144                           ArgAlaAlaAlaGluGlnLysLeuIleSerGluGluAspLeuAsnGly                              152025                                                                        GCCGCATAGACTGTTGAA162                                                         AlaAlaGluThrValGlu                                                            30                                                                            (2) INFORMATION FOR SEQ ID NO:133:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 112 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:133:                                     GlnValGlnLeuValGluSerGlyGlyGlyLeuValGlnProGlyGly                              151015                                                                        SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerSerTyr                              202530                                                                        SerMetAsnTrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal                              354045                                                                        SerTyrIleSerSerSerSerGlyThrIleTyrTyrAlaAspSerVal                              505560                                                                        LysGlyArgPheThrIleSerArgAspAsnAlaLysAsnSerLeuTyr                              65707580                                                                      LeuGlnMetAsnSerLeuArgAspGluAspThrAlaValTyrTyrCys                              859095                                                                        AlaSerSerSerTrpTyrGlyGlyTyrGlyAspTyrTrpGlyGlnGly                              100105110                                                                     (2) INFORMATION FOR SEQ ID NO:134:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 111 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:134:                                     GluValGlnLeuValGlnSerGlyGlyGlyLeuValGlnProGlyGly                              151015                                                                        SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerSerTyr                              202530                                                                        SerMetAsnTrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal                              354045                                                                        SerTyrIleSerSerSerSerSerThrIleTyrTyrAlaAspSerVal                              505560                                                                        LysGlyArgPheThrIleSerArgAspAsnAlaLysAsnSerLeuTyr                              65707580                                                                      LeuGlnMetAsnSerLeuArgAlaGluAspThrAlaValTyrTyrCys                              859095                                                                        AlaArgSerValAspSerTyrGlyMetAspValTrpGlyGlnGly                                 100105110                                                                     (2) INFORMATION FOR SEQ ID NO:135:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 120 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:135:                                     GlnValGlnLeuGlnGluSerGlyGlyGlyLeuValLysProGlyGly                              151015                                                                        SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerSerTyr                              202530                                                                        AlaMetHisTrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal                              354045                                                                        AlaValIleSerTyrAspGlySerAsnLysTyrTyrAlaAspSerVal                              505560                                                                        LysGlyArgPheThrIleSerArgAspAsnSerLysAsnThrLeuTyr                              65707580                                                                      LeuGlnMetAsnSerLeuArgAlaGluAspThrAlaValTyrTyrCys                              859095                                                                        AlaLysGlyGlyLeuGlyThrTyrTyrTyrAspSerSerGlyHisLys                              100105110                                                                     GlyPheAspProTrpGlyGlnGly                                                      115120                                                                        (2) INFORMATION FOR SEQ ID NO:136:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 101 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:136:                                     GlnValGlnLeuGlnGlnSerGlyProGlyLeuValLysProSerGln                              151015                                                                        ThrLeuSerLeuThrCysAlaIleSerGlyAspSerValSerSerAsn                              202530                                                                        SerAlaAlaTrpAsnTrpIleArgGlnTyrProSerArgGlyLeuGlu                              354045                                                                        TrpLeuGlyArgThrTyrTyrArgSerLysTrpTyrAsnAsnTyrAla                              505560                                                                        ValSerValLysSerArgIleThrIleAsnProAspThrSerLysAsn                              65707580                                                                      GlnPheSerLeuGlnLeuAsnSerValThrProGluAspLysAlaVal                              859095                                                                        TyrTyrCysAspLeu                                                               100                                                                           (2) INFORMATION FOR SEQ ID NO:137:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 101 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:137:                                     GlnValGlnLeuValGlnSerGlyProGlyLeuValLysProSerGln                              151015                                                                        ThrLeuSerLeuIleArgAlaIleSerGlyAspSerValSerSerAsn                              202530                                                                        SerAlaAlaTrpAsnTrpIleArgGlnSerProSerArgGlyLeuGlu                              354045                                                                        TrpLeuGlyArgThrTyrTyrArgSerLysTrpTyrAsnAspTyrAla                              505560                                                                        ValSerValLysSerArgIleIleIleAsnProGlyThrSerLysAsn                              65707580                                                                      GlnPheSerLeuGlnLeuSerSerValThrProGluAspThrAlaVal                              859095                                                                        TyrTyrCysAspLeu                                                               100                                                                           (2) INFORMATION FOR SEQ ID NO:138:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 101 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:138:                                     GlnValGlnLeuGlnGluSerGlyProGlyLeuValLysProSerGln                              151015                                                                        ThrLeuSerLeuThrCysAlaIleSerGlyAspSerValSerSerLys                              202530                                                                        SerAlaThrTrpAsnTrpIleArgGlnSerProSerArgGlyLeuGlu                              354045                                                                        TrpLeuGlyArgThrTyrTyrArgSerArgTrpTyrThrAspTyrAla                              505560                                                                        ThrSerValGlnSerArgIleThrIleAsnProAspThrSerLysAsn                              65707580                                                                      GlnPheSerLeuGlnLeuAsnSerMetThrProGluAspThrAlaVal                              859095                                                                        TyrTyrCysAspLeu                                                               100                                                                           (2) INFORMATION FOR SEQ ID NO:139:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 101 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:139:                                     GlnValGlnLeuGlnGlnSerGlyProGlyLeuMetLysProSerGln                              151015                                                                        ThrLeuSerLeuThrCysAlaIleSerGlyAspSerValSerSerSer                              202530                                                                        SerSerThrTrpAspTrpIleArgGlnSerProSerArgGlyLeuGlu                              354045                                                                        TrpLeuGlyArgThrTyrTyrArgSerLysTrpTyrAsnAspTyrAla                              505560                                                                        ValSerValLysSerArgIleThrIleLysAlaAspThrSerLysAsn                              65707580                                                                      GlnPheSerLeuGlnLeuSerSerValThrProGluAspThrAlaVal                              859095                                                                        TyrTyrCysAspPro                                                               100                                                                           (2) INFORMATION FOR SEQ ID NO:140:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 98 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:140:                                     GlnValAsnLeuArgGluSerGlyAlaGluValLysLysProGlyAla                              151015                                                                        SerValLysValSerCysLysAlaSerGlyPheThrPheThrSerSer                              202530                                                                        AlaValGlnTrpValArgGlnAlaProGlyGlnArgLeuGluTrpMet                              354045                                                                        GlyGlyIleIleProIlePheGlyThrAlaAsnTyrAlaGlnLysPhe                              505560                                                                        GlnGlyArgValThrIleThrAlaAspGluSerThrSerThrAlaTyr                              65707580                                                                      MetGluLeuSerSerLeuGlySerGluAspAlaAlaValTyrTyrCys                              859095                                                                        AspLeu                                                                        (2) INFORMATION FOR SEQ ID NO:141:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 116 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:141:                                     GlnValGlnLeuValGluSerGlyGlyGlyValValGlnProGlyArg                              151015                                                                        SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSerSerTyr                              202530                                                                        AlaMetHisTrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal                              354045                                                                        AlaValIleSerTyrAspGlySerAsnLysTyrTyrAlaAspSerVal                              505560                                                                        LysGlyArgPheThrIleSerArgAspAsnSerLysAsnThrLeuTyr                              65707580                                                                      LeuGlnMetAsnSerLeuArgAlaGluAspThrAlaValTyrTyrCys                              859095                                                                        AlaSerGlyArgTyrCysSerGlyGlySerCysSerProPheAspTyr                              100105110                                                                     TrpGlyGlnGly                                                                  115                                                                           (2) INFORMATION FOR SEQ ID NO:142:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 48 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:142:                                     MetLysTyrLeuLeuProThrAlaAlaAlaGlyLeuLeuLeuLeuAla                              22-20-15-10                                                                   AlaGlnProAlaMetAlaGlnValGlnLeuGlnValAspLeuGluIle                              51510                                                                         LysArgAlaAlaAlaGluGlnLysLeuIleSerGluGluAspLeuAsn                              152025                                                                        (2) INFORMATION FOR SEQ ID NO:143:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:143:                                     ValValProPheTyrSerGlnSerAlaGlnValGlnLeuGlnValAsp                              8-515                                                                         LeuGluIleLysArgAlaAlaAlaGluGlnLysLeuIleSerGluGlu                              101520                                                                        AspLeuAsnGlyAlaAlaGluThrValGlu                                                2530                                                                          (2) INFORMATION FOR SEQ ID NO:144:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 54 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:144:                                     ValLysLysLeuLeuPheAlaIleProLeuValValPheTyrAlaAla                              21-20-15-10                                                                   GlnProAlaMetAlaGlnValGlnLeuGlnValAspLeuGluIleLys                              51510                                                                         ArgAlaAlaAlaGluGlnLysLeuIleSerGluGluAspLeuAsnGly                              152025                                                                        AlaAlaGluThrValGlu                                                            30                                                                            __________________________________________________________________________

We claim:
 1. A method of making antibody polypeptide dimers specific foran antigen of interest, having the following steps:(i) providing nucleicacid expression vectors which are packaged using a component of areplicable genetic display package; (ii) combining (a) a geneticallydiverse repertoire of nucleic acid sequences which each encode a firstcomponent part of an antigen-binding site of a human antibody with (b)nucleic acid which encodes a unique or genetically diverse population ofa second component part of an antigen-binding site of a non-humanantibody known to bind said antigen of interest, to form a library ofnucleic acid sequences on said expression vectors encoding antibodypolypeptide dimers, which dimers each consist of a first polypeptidechain component and a second polypeptide chain component, in combinationforming an antigen-binding site of an antibody polypeptide dimer,wherein said library contains nucleic acid encoding an antibodypolypeptide dimer specific for an antigen of interest; (iii) expressingsaid library from said vectors in recombinant host organism cells, eachof the said first polypeptide chain components being expressed as afusion with a component of a replicable genetic display package whichthereby displays said first polypeptide chain component at the surfaceof replicable genetic display packages; (iv) selecting from saidexpressed library by binding with antigen a unique or restrictedpopulation of said antibody polypeptide dimers which have bindingspecificity for said antigen of interest, each selected antibodypolypeptide dimer being associated in its respective replicable geneticdisplay package with nucleic acid encoding said first component part ofthe antigen-binding site thereof.
 2. A method according to claim 1wherein said antibody polypeptide dimers are expressed as solublepolypeptides after selection in step (iv).
 3. A method according toclaim 1 wherein the sequence encoding each said second component part ofan antigen-binding site of a non-human antibody is modified by geneticalteration, said alteration is selected from the group consisting ofmutation, point mutation, insertion and deletion, to increase thehomology of said second component part of an antigen-binding site to asecond component part of a human antibody prior to said combining instep (ii).
 4. A method according to claim 1 having an additional step(v) wherein antibody polypeptide dimers selected in step (iv) aremodified by genetic alteration, said alteration is selected from thegroup consisting of mutation, point mutation, insertion and deletion, toremove or reduce non-human sequences.
 5. A method according to claim 1having an additional step (v) comprising:a) obtaining nucleic acidencoding said first component part from its replicable genetic displaypackage, displaying an antibody polypeptide dimer selected in step VIand combining the obtained nucleic acid with a genetically diverserepertoire of nucleic acid sequences each encoding a second componentpart of an antigen-binding site of human antibody, to form a secondlibrary of nucleic acid sequences encoding antibody polypeptide dimers,each antibody polypeptide dimer comprising a second antibody polypeptidechain component which is expressed from nucleic acid which is packagedusing a component of a replicable genetic display package, said secondchain component being expressed as a fusion with a component of areplicable genetic display package which thereby display said secondchain component at the surface of replicable genetic display packages,so that antibody polypeptide dimers specific for said antigen ofinterest, are selectable from said second library by binding with saidantigen of interest.
 6. A method according to claim 1 wherein saidsecond component part of an antigen-binding site of a non-human antibodyis an antibody region which binds an antigen.
 7. A method according toclaim 1 wherein said second component part of an antigen-binding site ofa non-human antibody is an antibody complementarity determining region.8. A method according to claim 1 wherein each antibody polypeptide dimerof any of said libraries of antibody polypeptide dimers is expressed asa single polypeptide chain.
 9. A method according to claim 1 whereineach antibody polypeptide dimer of any of said libraries of antibodypolypeptide dimers is expressed as two polypeptide chains.
 10. A methodof making human antibody polypeptide dimers specific for an antigen ofinterest, comprising:(i) combining a diverse population of polypeptideseach comprising a variable domain of a first polypeptide chain of ahuman antibody (population A) with a unique or restricted population ofpolypeptides each comprising a variable domain of a second polypeptidechain of a non-human antibody specific for said antigen (population B),thereby forming a library of antibody polypeptide dimers each consistingof a polypeptide which comprises a variable domain of a firstpolypeptide chain of a human antibody and a polypeptide chain of anon-human antibody, wherein said library contains an antibodypolypeptide dimer specific for an antigen of interest; (ii) selectingfrom said library a unique or restricted population of said antibodypolypeptide dimers which have binding specificity for said antigen(population c); (iii) combining a unique or restricted population ofhuman polypeptides derived from polypeptide dimers selected in step (ii)each comprising a human first polypeptide chain (population D) with adiverse population of polypeptides each comprising a variable domain ofa second polypeptide chain of a human antibody (population E), therebyforming a library of human antibody polypeptide chain dimers from whicha unique or restricted population chain dimers from which a unique orrestricted population of human antibody polypeptide dimers specific forsaid antigen (population F) are selectable.
 11. A method according toclaim 10 wherein:(a) said polypeptides of population A are eachexpressed from a vector (X) in recombinant host organism cells as afusion with a component of a replicable genetic display package whichthereby displays said polypeptide at the surface of replicable geneticdisplay packages in a first population of replicable genetic displaypackages; (b) nucleic acid of said vector (X) is packaged using saidreplicable genetic display package component, whereby the geneticmaterial of each said replicable genetic display package encodes a saidpolypeptide of population A, so that the antibody polypeptide dimers ofpopulation C are each associated in their respective replicable geneticdisplay package with nucleic acid encoding a polypeptide comprising avariable domain of a first polypeptide chain of a human antibody; (c)each of said polypeptides of population E is expressed from a vector (Y)in recombinant host organism cells as a fusion with a component of areplicable genetic display package which thereby displays it at thesurface of replicable genetic display packages in a second population ofreplicable genetic display packages; and (d) nucleic acid of said vector(Y) is packaged using said replicable genetic display package component,whereby the genetic material of each said replicable genetic displaypackage in the second population of replicable genetic display packagesencodes a said polypeptide of population E.
 12. A method according toclaim 11 wherein said population A is not expressed from the same vectoras said population B; and said population D is not expressed from thesame vector as said population E.
 13. A method according to claim 10wherein said population A is expressed from the same vector as saidpopulation B; and said population D is expressed from the same vector assaid population E.
 14. A method according to claim 13 wherein eachantibody polypeptide dimer of any of said libraries of antibodypolypeptide dimers is expressed as a single polypeptide chain.
 15. Amethod according to claim 11 wherein each antibody polypeptide dimer ofany of said libraries of antibody polypeptide dimers is expressed as twopolypeptide chains.
 16. A method according to claim 10 whereinpolypeptides of said population B are chimaeric, each comprising a humanantibody constant domain.
 17. A method according to claim 11 wherein thepolypeptides of said population E each comprise a region from anon-human antibody specific for said antigen, which region is one whichbinds antigen.
 18. A method according to claim 11 wherein thepolypeptides of said population E each comprise a complementaritydetermining region from a non-human antibody which specifically bindssaid antigen.
 19. A method according to claim 18 wherein said CDR isCDR3.
 20. A method according to claim 11 wherein said first polypeptidechain is antibody light chain, said second polypeptide chain beingantibody heavy chain.
 21. A method according to claim 11 comprising anadditional step of selecting said population F of human polypeptidedimers specific for said antigen.
 22. A method according to claim 11wherein any one of said populations C and F is selected by binding withsaid antigen.
 23. The method of claim 9 wherein said single polypeptidechain is a scFv fragment.
 24. The method of claim 10 wherein said twopolypeptide chains are selected from the group consisting of Fv and Fabfragments.
 25. The method of claim 15 wherein said single polypeptidechain is a scFv fragment.
 26. The method of claim 16 wherein said twopolypeptide chains are selected from the group consisting of Fv and Fabfragments.